Identification of heregulin, a specific activator of p185erbB2.

The proto-oncogene designated erbB2 or HER2 encodes a 185-kilodalton transmembrane tyrosine kinase (p185erbB2), whose overexpression has been correlated with a poor prognosis in several human malignancies. A 45-kilodalton protein heregulin-alpha (HRG-alpha) that specifically induced phosphorylation of p185erbB2 was purified from the conditioned medium of a human breast tumor cell line. Several complementary DNA clones encoding related HRGs were identified, all of which are similar to proteins in the epidermal growth factor family. Scatchard analysis of the binding of recombinant HRG to a breast tumor cell line expressing p185erbB2 showed a single high affinity binding site [dissociation constant (Kd) = 105 +/- 15 picomolar]. Heregulin transcripts were identified in several normal tissues and cancer cell lines. The HRGs may represent the natural ligands for p185erbB2.

Regulation of the acetylcholine receptor epsilon subunit gene by recombinant ARIA: an in vitro model for transynaptic gene regulation.

Structural specialization of the postsynaptic skeletal muscle membrane is in part mediated by the motor neuron-induced transcriptional regulation of synaptic muscle nuclei. ARIA, a factor that stimulates production of acetylcholine receptors (AChRs), is a candidate signaling molecule for such regulation. Here we examine the transynaptic inducing potential of this polypeptide factor. ARIA immunoreactivity is detectable at synaptic sites in vivo. In vitro, recombinant heregulin beta 1 (rHRG beta 1), the human homolog of ARIA, induces expression of the AChR epsilon gene, the subunit most sensitive to synaptic input. The inducing property of rHRG beta 1 is demonstrated most dramatically in primary muscle cultures from transgenic mice bearing an epsilon promoter-nuclear lacZ reporter transgene. Transient transfection experiments using the Sol 8 muscle cell line indicate that sequences that confer responsiveness to ARIA are located within a 150 bp epsilon subunit promoter region and are E box-independent. These results suggest that ARIA performs a vital role by directing spatially restricted gene expression at the neuromuscular junction.

A naturally occurring secreted human ErbB3 receptor isoform inhibits heregulin-stimulated activation of ErbB2, ErbB3, and ErbB4.

A variety of receptor-mediated signaling pathways are controlled by both positive and negative extracellular regulators. In this study, we demonstrate that a naturally occurring secreted form of the human ErbB3 receptor, p85-soluble ErbB3 (sErbB3), is a potent negative regulator of heregulin (HRG)-stimulated ErbB2, ErbB3, and ErbB4 activation. We show that p85-sErbB3 binds to HRG with an affinity comparable to that of full-length ErbB3 and competitively inhibits high affinity HRG binding to ErbB2/ErbB3 heterodimers on the cell surface of breast carcinoma cells with an IC(50) of 0.5 nM. p85-sErbB3 inhibits HRG-induced phosphorylation of ErbB2, ErbB3, and ErbB4 in breast carcinoma-derived cell lines and can also block HRG-stimulated activation of mitogen-activated protein kinase, Akt, and association of ErbB3 with the phosphatidylinositol 3'-kinase p85 regulatory subunit. Cell growth assays show that exogenous addition of a 100-fold molar excess of p85-sErbB3 inhibits HRG-stimulated cell growth by as much as 90%. Whereas several potential mechanisms of p85-sErbB3 inhibition of ErbB receptor activation exist, our results suggest that at least one means of inhibition is competition for HRG binding. The IC(50) for both p85-sErbB3- and 2C4 (a monoclonal antibody specific for ErbB2)-mediated inhibition of HRG binding is approximately 0.5 nM, although the mechanism of inhibition by these two proteins is distinct. Together these results suggest that p85-sErbB3 is a naturally occurring negative regulator of HRG-stimulated signal transduction that may have important therapeutic applications in human malignancies associated with HRG-mediated cell growth such as breast and prostate cancer.

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.

Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells.

HER2/neu (erbB-2) overexpression has been causally associated with tamoxifen resistance in human breast cancer cells. Forced expression of HER2 in MCF-7 breast cancer cells resulted in mitogen-activated protein kinase (MAPK) hyperactivity and tamoxifen resistance. Inhibition of HER2 and MAPKs with AG1478 and U0126, respectively, as well as dominant-negative MEK-1/2 constructs restored the inhibitory effect of tamoxifen on estrogen receptor (ER)-mediated transcription and cell proliferation. Both AG1478 and U0126 also restored the tamoxifen-mediated association of ER with nuclear receptor corepressor (N-CoR) in the antiestrogen-resistant MCF-7 cells. Treatment with a combination of tamoxifen and a HER2 kinase inhibitor reduced tumor MAPK activity and markedly prevented growth of HER2-overexpressing MCF-7 xenografts in athymic mice. Thus, blockade of HER2 and MAPK signaling may enhance tamoxifen action and abrogate antiestrogen resistance in human breast cancer.

Gamma-heregulin: a novel heregulin isoform that is an autocrine growth factor for the human breast cancer cell line, MDA-MB-175.

A novel neuregulin isoform, termed gamma-HRG, was cloned and characterized from the human breast cancer cell line, MDA-MB-175. As observed with other neuregulins, gamma-HRG, is a product of alternative mRNA splicing of the neuregulin gene. Gamma-HRG contains the EGF-like and immunoglobulin-like domains that are commonly found in other family members, but lacks a transmembrane and cytoplasmic region. The new isoform possesses a unique N-terminal region that includes a hydrophobic domain that may function as a secretion signal. A purified recombinant version of gamma-HRG competes for binding to soluble ErbB3- and ErbB4-IgG fusion proteins with affinities similar to those observed for rHRGbeta1(177-244). Gamma-HRG has a wide distribution in mesenchymal or neuronal tissues but in contrast to other neuregulins, it is not present in breast, lung, liver and small intestine. Expression of gamma-HRG with its cognate receptors, ErbB3 and ErbB2 suggested that the growth of the MDA-MB-175 cell line might be a result of the autocrine stimulation of a growth factor signaling pathway. Treatment of MDA-MB-175 cells with an anti-ErbB2 monoclonal antibody that interferes with the ligand-dependent formation of ErbB2-ErbB3 heterodimer complexes shows a strong growth inhibitory effect on this cell line. Moreover, incubation with a receptor-IgG fusion protein that neutralizes secreted gamma-HRG, also inhibits cell growth. These data suggest that the secretion of gamma-HRG by MDA-MB-175 cells leads to the formation of a constitutively active receptor complex and stimulates the growth of these cells in an autocrine manner.

Heregulin is rapidly translocated to the nucleus and its transport is correlated with c-myc induction in breast cancer cells.

Heregulins (neuregulins) are a family of proteins known to interact and activate the receptor tyrosine kinases ErbB2 in association with ErbB3 or ErbB4. Using immunofluorescence microscopy, electron microscopy autoradiography, and SDS-PAGE analysis of nuclear fractions, we show that the heregulin-beta1(1-244) isoform is rapidly internalized and translocated to the nucleus of SK-BR-3 breast cancer cells as an intact molecule. Heregulin-beta 1(1-244) treatment up-regulated expression of c-myc mRNA and protein, which was also observed to undergo its own translocation from the cytosol to the nucleus. c-myc thus appears to be a cellular target gene of HRGbeta 1(1-244), and its induction may be related to the nuclear translocation of heregulin.

HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells.

Growth of human breast cells is closely regulated by steroid hormone as well as peptide hormone receptors. Members of both receptor classes are important prognostic factors in human breast cancer. Clinical data indicate that overexpression of the HER-2 gene is associated with an estrogen receptor-negative phenotype. In this study, we demonstrate that introduction of a HER-2 cDNA, converting non-overexpressing breast cancer cells to those which overexpress this receptor, results in development of estrogen-independent growth which is insensitive to both estrogen and the antiestrogen, tamoxifen. Moreover, activation of the HER-2 receptor in breast cancer cells by the peptide growth factor, heregulin, leads to direct and rapid phosphorylation of ER on tyrosine residues. This is followed by interaction between ER and the estrogen-response elements in the nucleus and production of an estrogen-induced protein, progesterone receptor. In addition, overexpression of HER-2 receptor in estrogen-dependent tumor cells promotes ligand-independent down-regulation of ER and a delayed autoregulatory suppression of ER transcripts. These data demonstrate a direct link between these two receptor pathways and suggest one mechanism for development of endocrine resistance in human breast cancers.

Biologic effects of heregulin/neu differentiation factor on normal and malignant human breast and ovarian epithelial cells.

The heregulins are a family of ligands with ability to induce phosphorylation of the p185HER-2/neu receptor. Various investigators have reported a variety of responses of mouse and human breast and ovarian cells to this family of ligands including growth stimulation, growth inhibition, apoptosis and induction of differentiation in cells expressing the HER-2/neu receptor. Some of the disparity in the literature has been attributed to variations in the cell lines studied, ligand dose applied, methodologies utilized or model system evaluated (i.e. in vitro or in vivo). To evaluate the effects of heregulin on normal and malignant human breast and ovarian epithelial cells expressing known levels of the HER-2/neu receptor, this report presents the use of several different assays, performed both in vitro and in vivo, in vitro proliferation assays, direct cell counts, clonogenicity under anchorage-dependent and anchorage-independent conditions, as well as the in vivo effects of heregulin on human cells growing in nude mice to address heregulin activity. Using a total of five different biologic assays in nine different cell lines, across two different epithelia and over a one log heregulin dose range, we obtained results that clearly indicate a growth-stimulatory role for this ligand in human breast and ovarian epithelial cells. We find no evidence that heregulin has any growth-inhibitory effects in human epithelial cells. We also quantitated the amount of each member of the type I receptor tyrosine kinase family (RTK I, i.e. HER-1, HER-2, HER-3 and HER-4) in the cell lines employed and correlated this to their respective heregulin responses. These data demonstrate that HER-2/neu overexpression itself affects the expression of other RTK I members and that cells expressing the highest levels of HER-2/neu have the greatest response to HRG.

Backbone dynamics of the EGF-like domain of heregulin-alpha.

The backbone dynamics of the 63 residue epidermal growth factor (EGF)-like domain of heregulin-alpha (HRG-alpha) have been characterized by measurement of longitudinal relaxation rate constants (R1), transverse relaxation rate constants (R2), and steady-state ¿1H¿-15N nuclear Overhauser effects for the 15N nuclear spins using proton-detected heteronuclear NMR spectroscopy. Analysis of the R2/R1 ratios in conjunction with the known structure of the HRG-alpha EGF-like domain yields a rotational correlation time of approximately 8.4 ns, suggesting that the protein aggregates under the solution conditions used (3.8 mM protein, 50 mM sodium acetate, pH 4.5, 20 degreesC), and that it tumbles with an axially symmetric diffusion tensor (D parallel/D perpendicular=1.4). Sedimentation equilibrium experiments confirm that the EGF-like domain of HRG-alpha undergoes weak self-association under these conditions and are consistent with a simple monomer-dimer equilibrium with a dimer-dissociation constant Kd=1.6(+/-0.4) mM. The relaxation data were analyzed using a reduced spectral density mapping approach to avoid systematic effects of aggregation on the usual model-free formalism. The reduced spectral densities show that residues near the N terminus (residues 3 to 5 and 7 to 12), in the Omega-loop between beta-strands 2 and 3 (residues 24 to 31), and in particular the C-terminal 13 residues (residues 51 to 63), have significant mobility on a picosecond/nanosecond time-scale. In addition, conformational exchange on a microsecond time-scale was identified for residues 44 to 46 on the basis of observed differences in R2 at 11.7 and 14.1 T. The mobility identified near the N terminus and in the vicinity of residues 44 to 46 may be important in allowing the interactions of heregulin with multiple receptors.

Biological response to ErbB ligands in nontransformed cell lines correlates with a specific pattern of receptor expression.

The human epidermal growth factor receptor (HER or ErbB) family consists of four distinct members, including the epidermal growth factor (EGF) receptor (EGFR, HER1, or ErbB1), ErbB2 (HER2 or neu), ErbB3 (HER3), and ErbB4 (HER4). Activation of these receptors plays an important role in the regulation of cell proliferation, differentiation, and survival in several different tissues. Binding of a specific ligand to one of the ErbB receptors triggers the formation of specific receptor homo- and heterodimers, with ErbB2 being the preferred signaling partner. We analyzed the levels of various ErbB receptor messenger RNAs in a series of nontransformed cell lines by real time quantitative RT-PCR. The cell lines chosen were derived from a variety of tissues, including pancreas, lung, heart, and nervous system. Further, we measured biological responses in these cell lines upon treatment with EGF, betacellulin, and two types of neuregulins, heregulin and sensory and motor neuron-derived factor. All cell lines examined expressed detectable levels of ErbB2. High levels of expression of ErbB3 were correlated with responsiveness to heregulin and sensory and motor neuron-derived factor, whereas high levels of EGFR expression were correlated with responsiveness to EGF and betacellulin. Moreover, the sensitivity of a cell line to ErbB ligands was also correlated with the levels of expression of the appropriate ErbB receptors in that cell line. These results are consistent with our hypothesis that appropriate biological responsiveness to ErbB ligands is determined by the levels of expression of specific ErbB receptor combinations within a given tissue.

Binding specificities and affinities of egf domains for ErbB receptors.

ErbB receptor activation is a complex process and is dependent upon the type and number of receptors expressed on a given cell. Previous studies with defined combinations of ErbB receptors expressed in mammalian cells have helped elucidate specific biological responses for many of the recognized gene products that serve as ligands for these receptors. However, no study has examined the binding of these ligands in a defined experimental system. To address this issue, the relative binding affinities of the egf domains of eleven ErbB ligands were measured on six ErbB receptor combinations using a soluble receptor-ligand binding format. The ErbB2/4 heterodimer was shown to bind all ligands tested with moderate to very high affinity. In contrast, ErbB3 showed much more restrictive ligand binding specificity and measurable binding was observed only with heregulin, neuregulin2beta, epiregulin and the synthetic heregulin/egf chimera, biregulin. These studies also revealed that ErbB2 preferentially enhances ligand binding to ErbB3 or ErbB4 and to a lesser degree to ErbB1.

Formation of a high affinity heregulin binding site using the soluble extracellular domains of ErbB2 with ErbB3 or ErbB4.

ErbB2 functions as a shared signal transducing component for other ErbB receptor family members. Two of these receptors, ErbB3 and ErbB4, bind the heregulin (HRG) or neuregulin family of polypeptide growth factors. Cells expressing ErbB3 alone display a single class of low affinity HRG binding sites, whereas both high and low affinity binding sites can be measured on cells that co-express both ErbB3 and ErbB2. To assess the interaction of the extracellular domains of ErbB receptors, a series of soluble homodimeric and heterodimeric IgG fusion proteins were constructed. Heregulin binding analysis revealed that a heterodimer composed of either ErbB3 or ErbB4 with ErbB2 is sufficient for the formation of a high affinity binding state. In contrast, heterodimeric ErbB3/4-IgG, as well as homodimeric ErbB3-IgG or ErbB4-IgG, contained only low affinity HRG binding sites. Further evidence for the unique specificity of ErbB2 in generating this high affinity binding site was determined by inhibiting HRG binding with an ErbB2 monoclonal antibody.

Neuregulin receptor-mediated gene transfer by human epidermal growth factor receptor 2-targeted antibodies and neuregulin-1.

The human epidermal growth factor receptors 2, 3, and 4 (HER2, HER3, and HER4, respectively) are frequently overexpressed in many human cancers, and therefore may be potential targets for receptor-mediated gene transfer. To evaluate this possibility, we constructed a series of HER-targeted gene transfer vehicles by covalently linking poly-L-lysine polymers (pLYS) to the epidermal growth factor-like domain of the HER ligand neuregulin-1 (NRG1(177-244)), a HER2 antibody (Ab), and the Fab fragment of the HER2 Ab. In vitro, pLYS modification of NRG1(177-244) decreased the affinity of the ligand for HER3 or HER4 homodimer receptors by 6- to 7-fold. DNA loading of the pLYS-modified NRG1(177-244) had a minimal additional affect on the affinity of the complex for its receptor. In cell lines engineered to solely express HER2, HER3, or HER4, each vehicle correctly targeted the receptors; the NRG1(177-244) construct transferred a luciferase gene only into cells expressing HER4, whereas the HER2 Ab and Fab constructs transferred the reporter gene only into cells expressing HER2. The most efficient gene transfer occurred using the intact HER2 Ab as a gene transfer vehicle, whereas the Fab fragment of the HER2 Ab was the least efficient, and NRG1(177-244) was intermediate. These studies suggest that the NRG receptor or HER2, a component of the receptor, can be pursued as targets for gene transfer.

CD20: a gene in search of a function.

CD20 is a signature B-cell differentiation antigen. The function of CD20 is unknown, although it is thought to be involved in B-cell activation, regulation of B-cell growth, and transmembrane calcium flux. This review covers several topics important for understanding CD20 biology. These topics include the expression pattern and transcriptional regulation of the CD20 gene, the structure of the protein and its interaction with other cell surface molecules, as well as CD20 phosphorylation and putative functions. An understanding of CD20 function from signal transduction to biological implications may prove important for the mechanistic understanding of the treatment of certain types of cancer.

Structural requirements for ErbB2 transactivation.

ErbB2 is a unique member of the ErbB family of receptor tyrosine kinases that is distinguished by the fact that no ligand has yet been identified. Due to the absence of an ErbB2 ligand, alternative mechanisms are used for ErbB2 activation. As such, when ErbB2 is overexpressed, kinase activation occurs in the absence of ligand because of constitutive homodimerization. However, at normal expression levels ErbB2 acts as the shared coreceptor for the ErbB family, and these heterodimeric complexes are activated in response to the partner ligand. While the extracellular domain and transmembrane domains are necessary for ErbB2 transactivation, the carboxy terminus is also required. Specifically, ligand-dependent ErbB2 transactivation requires a discrete three-amino-acid segment, located at the C-terminus of ErbB family members ErbB3, ErbB4, and the epidermal growth factor receptor. Transactivation of ErbB2 via the three-amino-acid segment likely represents a conserved mechanism for regulated signaling by the ErbB family of receptors.

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.

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.

Establishment of Schwann cell lines from normal adult and embryonic rat dorsal root ganglia.

Schwann cells, an important component of the peripheral nervous system, interact with neurons to mutually support growth and replication in the embryo and survival and differentiated function in the adult. The ability of adult Schwann cells to re-enter the cell cycle after nerve injury is crucial to their role in nerve repair. This ability suggests that it should be possible to obtain non-transformed, cell lines which maintain the characteristics of proliferating adult Schwann cells in vivo, as well as obtaining Schwann cells from rapidly dividing embryonic tissues. One approach to obtaining normal functionally differentiated cell lines has been to start primary cultures in serum-free medium containing growth factors and attachment proteins specifically selected to favor the replication of the cell type of interest. By culturing dispersed dorsal root ganglia on laminin, in serum-free medium with hormones and growth factors, we repeatedly generate homogenous Schwann cell cultures which yield normal Schwann cell lines from the dorsal root ganglia (DRG) of both embryonic and adult rats. These cells maintain the phenotype of Schwann cells as determined by morphology and staining for GFAP, S100, p75 NGF receptor, laminin, and MAG production in co-culture with DRG neurons.

Selenium-dependent glycine reductase: differences in physicochemical properties and biological activities of selenoprotein A components isolated from Clostridium sticklandii and Clostridium purinolyticum.

The selenoprotein A component of the glycine reductase complex of Clostridium sticklandii was shown to differ in certain properties from the selenoprotein A produced by a purine-fermenting organism, Clostridium purinolyticum. Both proteins contain one selenocysteine and two cysteine residues.