A potential role for activated HER-2 in prostate cancer.

The epidermal growth factor (also known as HER or ErbB) family of receptor tyrosine kinases are important mediators of cell growth, differentiation, and survival. At present there are 10 ligands that bind directly to epidermal growth factor, HER-3, or HER-4. Although none of these ligands bind directly to HER-2, it is recruited to these receptor complexes and also becomes activated. A monoclonal antibody directed against HER-2, 2C4, inhibits the association of HER-2 with other HER family members. Ligand-activated HER-2 may also play a role in cancers, particularly those that do not overexpress HER-2 at high levels. For example, when prostate cancers progress from an androgen-dependent to an androgen-independent phenotype, epidermal growth factor pathways are frequently activated. 2C4 will inhibit the growth of both androgen-dependent and androgen-independent prostate tumors grown as xenografts in athymic mice.

Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin).

HER2 is a ligand-less member of the human epidermal growth factor receptor or ErbB family of tyrosine kinases. In normal biological systems, HER2 functions as a co-receptor for a multitude of epidermal growth factor-like ligands that bind and activate other HER family members. HER2 overexpression is observed in a number of human adenocarcinomas and results in constitutive HER2 activation. Specific targeting of these tumors can be accomplished with antibodies directed against the extracellular domain of the HER2 protein. One of these antibodies, 4D5, has been fully humanized and is termed trastuzumab (Herceptin; Genentech, San Francisco, CA). Treatment of HER2-overexpressing breast cancer cell lines with trastuzumab results in induction of p27KIP1 and the Rb-related protein, p130, which in turn significantly reduces the number of cells undergoing S-phase. A number of other phenotypic changes are observed in vitro as a consequence of trastuzumab binding to HER2-overexpressing cells. These phenotypic changes include downmodulation of the HER2 receptor, inhibition of tumor cell growth, reversed cytokine resistance, restored E-cadherin expression levels, and reduced vascular endothelial growth factor production. Interaction of trastuzumab with the human immune system via its human immunoglobulin G1 Fc domain may potentiate its antitumor activities. In vitro studies demonstrate that trastuzumab is very effective in mediating antibody-dependent cell-mediated cytotoxicity against HER2-overexpressing tumor targets. Trastuzumab treatment of mouse xenograft models results in marked suppression of tumor growth. When given in combination with standard cytotoxic chemotherapeutic agents, trastuzumab treatment generally results in statistically superior antitumor efficacy compared with either agent given alone. Taken together, these studies suggest that the mechanism of action of trastuzumab includes antagonizing the constitutive growth-signaling properties of the HER2 system, enlisting immune cells to attack and kill the tumor target, and augmenting chemotherapy-induced cytotoxicity.

Binding interaction of the heregulinbeta egf domain with ErbB3 and ErbB4 receptors assessed by alanine scanning mutagenesis.

Individual residues of the heregulinbeta (HRG) egf domain were mutated to alanine and displayed monovalently on phagemid particles as gene III fusion proteins. Wild type HRGbeta egf domain displayed on phage was properly folded as evidenced by its ability to bind ErbB3 and ErbB4 receptor-IgG fusion proteins with affinities close to those measured for bacterially produced HRGbeta egf domain. Binding to ErbB3 and ErbB4 receptors was affected by mutation of residues throughout the egf domain; including the NH2 terminus (His2 and Leu3), the two beta-turns (Val15-Gly18 and Gly42-Gln46), and some discontinuous residues (including Leu3, Val4, Phe13, Val23, and Leu33) that form a patch on the major beta-sheet and the COOH-terminal region (Tyr48 and Met50-Phe53). Binding affinity was least changed by mutations throughout the Omega-loop and the second strand of the major beta-sheet. More mutants had greater affinity loss for ErbB3 compared with ErbB4 implying that it has more stringent binding requirements. Many residues important for HRG binding to its receptors correspond to critical residues for epidermal growth factor (EGF) and transforming growth factor alpha binding to the EGF receptor. Specificity may be determined in part by bulky groups that prevent binding to the unwanted receptor. All of the mutants tested were able to induce phosphorylation and mitogen-activated protein kinase activation through ErbB4 receptors and were able to modulate a transphosphorylation signal from ErbB3 to ErbB2 in MCF7 cells. An understanding of binding similarities and differences among the EGF family of ligands may facilitate the development of egf-like analogs with broad or narrow specificity.

Selection of heregulin variants having higher affinity for the ErbB3 receptor by monovalent phage display.

Heregulins (HRGs) are epidermal growth factor (egf) domain containing polypeptide growth factors that bind and activate several members of the ErbB receptor family. Although HRG can bind to ErbB3 and ErbB4 homodimers, the highest affinity and most intracellularly active receptor complexes are hetero-oligomers containing ErbB2. The HRGbeta egf domain was displayed on the surface of M13 phage to facilitate mutagenic analysis and optimize for binding to a homodimeric ErbB3-immunoglobulin (IgG) fusion. Nine libraries were constructed in which virtually the entire sequence was randomized in stretches of four to six amino acids. These were selected separately for binding to immobilized ErbB3-IgG. Analysis of the resulting sequences revealed some areas that diverged radically from the wild-type, whereas others showed strong conservation. The degree of wild-type conservation correlated strongly with the functional importance of the residues as determined by alanine scanning mutagenesis (Jones, J. T., Ballinger, M. D., Pisacane, P. I., Lofgren, J. A., Fitzpatrick, V. D., Fairbrother, W. J., Wells, J. A., and Sliwkowski, M. X. (1998) J. Biol. Chem. 273, 11667-11674). Some variants from several libraries showed significant improvements in binding affinity to the ErbB3-IgG. These optimized segments were combined in various ways in the same molecule to generate variants (containing up to 16 mutations) that had >50-fold higher affinity than wild-type HRGbeta. The optimized variants stimulated ErbB2 phophorylation on MCF7 cells at levels similar to wild-type. This indicates wild-type affinity is optimized for potency and that factors other than affinity for ErbB3 are limiting. These variants showed enhanced affinity toward the ErbB4 homodimer, suggesting these receptors use very similar binding determinants despite them having 65% sequence identity.

Analysis of heregulin-induced ErbB2 phosphorylation with a high-throughput Kinase receptor activation enzyme-linked immunosorbant assay.

A rapid, sensitive, and high-throughput assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a kinase receptor activation enzyme-linked immunosorbant assay (KIRA-ELISA), consists of two separate microtiter plates, one for cell culture, ligand stimulation, and cell lysis/receptor solubilization and the other plate for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of heregulin-induced ErbB2 activation and utilizes the stimulation of intact receptor on the adherent breast carcinoma cell line, MCF-7. Membrane proteins are solubilized via Triton X-100 lysis and the receptor is captured in ELISA wells coated with ErbB2-specific antibodies with no cross-reaction to ErbB3 or ErbB4. The degree of receptor phosphorylation is then quantified by antiphosphotyrosine ELISA. A reproducible standard curve is generated with a EC(50) of approximately 360 pM for heregulin beta 1(177-244) (HRG beta 1(177-244). When identical samples of HRG beta 1(177-244) are analyzed by both the KIRA-ELISA and quantitative antiphosphotyrosine Western blot analysis, the results correlate very closely with one another. The assay described in this report is able to specifically quantify tyrosine phosphorylation of ErbB2 that results from the interaction of HRG with ErbB3 and/or ErbB4.

Growth regulation of human breast and ovarian tumor cells by heregulin: Evidence for the requirement of ErbB2 as a critical component in mediating heregulin responsiveness.

Alterations in the expression of the epidermal growth factor (EGF) receptor ErbB family are frequently encountered in a number of human cancers. Two of these receptors, ErbB3 and ErbB4, are known to bind a family of related proteins termed heregulins (HRGs) or neu differentiation factors. In biologically relevant systems, interaction of HRG with ErbB3 or ErbB4 results in the transactivation of ErbB2. In this report, we show that ErbB2 is a critical component in mediating HRG responsiveness in a panel of human breast and ovarian tumor cell lines. Because HRGs have been reported to elicit diverse biological effects on cultured cells, including growth stimulation, growth inhibition, and induction of differentiation, we systematically examined the effect of rHRG beta 1 on tumor cell proliferation. HRG binding studies were performed with a panel of breast and ovarian tumor cell lines expressing a range of levels of ErbB2. The biological responses to HRG were also compared to EGF and to the growth-inhibitory anti-ErbB2 antibody, 4D5. In most cases, HRG stimulation of DNA synthesis correlated with positive effects on cell cycle progression and cell number and with enhancement of colony formation in soft agar. On each cell line tested, the HRG effects were distinguishable from EGF and 4D5. Our findings indicate that HRG induces cell proliferation in a number of tumor cell lines. In addition, we show that methods for measuring cell proliferation, as well as experimental conditions, are critical for determining HRGs effect on tumor cell growth in vitro.

The influence of heregulins on human Schwann cell proliferation.

The use of Schwann cell (SC) autotransplantation to influence neural repair in humans is dependent upon identifying mitogens that will effectively expand human Schwann cells (SCs) in culture. The recent purification and molecular cloning of glial growth factor (GGF), a potent mitogen for rat Schwann cells, has led to the recognition that a family of proteins (GGF/HRG/NDF/ARIA) are alternatively spliced products of a single gene. The heregulins (HRGs) have been characterized with respect to their influence on human breast cancer cell lines; here we examined whether the HRGs have mitogenic activity for human SCs. Using DNA synthesis assays and serial passaging of cells in culture, we demonstrate that HRG is an effective mitogen for human SCs and that, in the presence of agents that elevate cAMP, it is possible to expand these cells over multiple passages without overwhelming fibroblast contamination. One putative target for this family of proteins is p185erbB2, and EGF-like receptor tyrosine kinase that is encoded by the erbB2 protooncogene. In this report we also demonstrate that the erbB2/3/4 messages as well as the erbB2/3 receptor proteins are present within cultured human SCs. The addition of HRG to human SCs results in tyrosine phosphorylation of a 185 kDa protein. In the presence of stimulatory concentrations of HRG, a blocking monoclonal antibody (2C4) to p185erbB2 is capable of significantly inhibiting phosphorylation of a 185 kDa protein as well as the subsequent incorporation of 3H-thymidine within the human SC. These latter results implicate an important role for p185erbB2 in mediating the mitogenic response of human SCs to HRGs.

Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin.

The heregulin/neu differentiation factor gene products were purified and cloned based on their ability to stimulate the phosphorylation of a 185-kDa protein in human breast carcinoma cell lines known to express erbB2. However, not all cells that express erbB2 respond to heregulin, indicating that other components besides erbB2 may be required for heregulin binding. Cells that are transfected with the closely related receptor, erbB3, display a single class of lower affinity heregulin binding sites than has been previously observed on breast carcinoma cell lines. Little or no stimulation of tyrosine phosphorylation in response to heregulin occurs in cells that are transfected with erbB3 alone. Transfection of cells with erbB3 and erbB2 reconstitutes a higher affinity binding receptor, which is also capable of generating a tyrosine phosphorylation signal in response to heregulin. A monoclonal antibody to erbB2 will inhibit heregulin activation of tyrosine phosphorylation and binding in cells transfected with both receptors but not with erbB3 alone. In cells expressing erbB2 and erbB3, both proteins become tyrosine-phosphorylated upon interaction with heregulin. Direct interaction between heregulin and the two proteins was demonstrated by chemical cross-linking experiments using 125I-heregulin followed by immunoprecipitation with antibodies specific for erbB2 or erbB3.

Monoclonal antibody based ELISAs for measurement of activins in biological fluids.

Two sensitive monoclonal antibody (MAb)-based enzyme-linked immunosorbent assays (ELISAs), one for activin A (homodimer of beta A subunits) and one for activin B (homodimer of beta B subunits) in plasma have been developed. The activin A ELISA had an effective range of 0.2-50 ng/ml while the activin B ELISA's range was 0.1-25 ng/ml in human serum. Both ELISAs were specific with < 0.01% cross-reactivity with related hormones and follistatin (an activin binding protein), however the presence of recombinant human follistatin caused a decrease in measured level of activin A and B spiked human samples. The assay was linear across the standard curve range with intra- and interassay coefficients of variation were less than 15%. The level of activins in female serum range from 0.3 to 10.4 ng/ml. In summary, we have developed a reliable, convenient and rapid MAb-based enzyme immunoassay for determination of activin A and B levels in human serum which are also applicable for buffer, mouse and monkey serum matrices. This assay will be useful for studying the regulation and role of activin A and B in health and disease.

Relaxin binding in the rat heart atrium.

Relaxin is a member of the insulin family of polypeptides that is best known as a reproductive hormone. In an effort to elucidate the mechanism of action of relaxin we previously localized the specific binding sites of a 32P-labeled relaxin in the rat uterus and brain. These studies suggested that, in addition to its classical role in pregnancy, relaxin might have other physiological functions. In the present paper we describe the specific and high-affinity binding of relaxin to the cardiac atrium of both male and female rats. The relaxin binding could not be displaced by peptides belonging to the same family [insulin, insulin-like growth factor I (IGF-I)] or by peptides that were identified in the atrium or were known to have cardiovascular functions (atrial natriuretic peptide, angiotensin II). The dissociation constant for relaxin in the atrium was estimated to be 1.4 nM, which was similar to that found in the uterus (1.3 nM) and the brain (1.4 nM). In view of the close association of relaxin with reproduction, an experiment was also performed to compare the relaxin binding in the uterus and heart after gonadectomy and sex steroid treatment. It was found that the relaxin binding in the rat uterus was diminished by 53% overall following ovariectomy but was restored to 90% of normal levels when treated with estrogen (but not with testosterone). In contrast, the relaxin binding in the rat heart was not affected by castration or sex steroid treatment. We conclude that specific and high-affinity relaxin receptors exist in the atrium of both the male and female rat heart and that these are regulated differently than the relaxin receptors in the uterus.

In vivo regulation of FSH synthesis by inhibin and activin.

The effect of a single sc injection of the gonadal peptide, recombinant human activin A (rhActivin A), on gonadotropin synthesis and secretion was examined in adult and immature male and female rats and the effect of recombinant human inhibin A (rhInhibin A) was examined in adult male rats. Pituitary FSH beta, LH beta and alpha messenger RNA (mRNA) levels were determined by blot hybridization. Trunk blood was collected to measure serum FSH levels. Treatment with rhInhibin A (100 micrograms/kg) resulted in a decrease in FSH beta mRNA to 2% of controls levels 6 h after injection. FSH beta mRNA levels started to rebound at 10 h, but were still significantly lower than vehicle-treated controls. Serum FSH levels were significantly reduced at 2 h and were reduced further at 6 and 10 h. There were no significant changes in alpha and LH beta mRNA levels. RhActivin A, at the highest dose (500 micrograms/kg), in immature male rats had only a modest effect (1.2- and 1.3-fold increase) on FSH beta mRNA levels and FSH secretion, respectively, at 2 h. No increase in FSH synthesis and FSH secretion was observed in adult male rats. In contrast, both immature and adult-ovariectomized E2 implanted females showed a robust response to rhActivin A. In immature females, 2 h after rhActivin A (100 and 500 micrograms/kg) administration, FSH beta mRNA levels were elevated 2.0- and 2.2-fold. At this time serum FSH was also elevated. At 6 and 10 h rhActivin A significantly reduced FSH beta mRNA levels from vehicle-treated controls. In contrast, FSH secretion was elevated at 6 h and returned to baseline at 10 h. Administration of rhActivin A (500 micrograms/kg) to adult, ovariectomized-E2 females resulted in a significant increase in FSH beta mRNA levels and FSH secretion at 2 and 6 h. There were no significant changes in alpha and LH beta mRNA levels in either males or females. Thus, these in vivo studies have shown that rhInhibin A can inhibit FSH beta mRNA levels and FSH secretion in the adult male rat. RhActivin A stimulates FSH synthesis and secretion in the immature and adult ovariectomized-E2 females, but has little or no effect in immature and adult males. Hence, there is a sexual dimorphic response to rhActivin A in vivo in the rat.

Generation of polyclonal antibodies against recombinant human activin A.

A goat antiserum to purified recombinant human activin A (rhAct-A), a dimer formed by two beta A-subunits of inhibin, has been produced. The immunoreactivity of the antiserum has been evaluated in an antigen coated enzyme-linked immunosorbent assay, in a radioimmunoassay using iodinated rhAct-A, and by Western blot analysis. The antiserum demonstrated some cross reactivity to inhibin A, a structurally related heterodimer which contains an identical beta A-subunit coupled to a distinct, though similar, alpha subunit. A simple radioimmunoassay for rhAct-A in tissue culture supernatant has been developed with rhAct-A affinity column purified polyclonal antiserum. The assay is precise and sensitive with a range of 0.31-40 ng/ml. The cross reactivity of inhibin A in the RIA is about 4.3%. Despite its cross-reactivity this antiserum will facilitate studies of the physiology of activin A and inhibin A which includes a Western blot analysis where a molecular size distinction is accomplished.

Preparation of biologically active 32P-labeled human relaxin. Displaceable binding to rat uterus, cervix, and brain.

Relaxin is a member of the insulin family of polypeptide hormones and is known to exert its biological effects on various parts of the mammalian reproductive system. Biologically active human relaxin has been chemically synthesized based on the nucleotide sequence obtained from an ovarian cDNA clone. In the present study synthetic human relaxin was radiolabled by phosphorylation with cAMP-dependent protein kinase and [gamma-32P]ATP to a specific activity of 5000 Ci/mmol. The phosphorylated relaxin was purified on cation exchange high performance liquid chromatography and was shown to co-migrate with relaxin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed a single phosphorylated site on the B chain of relaxin. The 32P-relaxin was able to bind to a goat anti-relaxin antibody, and this binding could be displaced by unlabeled relaxin in a concentration-dependent manner. A comparison of the concentration responses of cellular cAMP production stimulated by relaxin and phosphorylated relaxin in a primary human uterine cell line showed that phosphorylation did not affect the in vitro biological efficacy of relaxin. This made it suitable for in situ autoradiographic localization of relaxin binding sites in rat uterine, cervical, and brain tissue sections. Displacement of the binding of 100 pM 32P-relaxin by 100, 10, and 3 nM unlabeled relaxin, but not by 100 nM insulin, insulin-like growth factor-I, and an insulin-like growth factor-I analog, demonstrated the high affinity and specificity of such binding. We conclude that 32P-labeled human relaxin is biologically and immunologically active and that this novel probe binds reversibly and with high affinity to classical (e.g. uterus) and unpredicted (e.g. brain) tissues.