ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling.

We have analyzed ErbB receptor interplay induced by the epidermal growth factor (EGF)-related peptides in cell lines naturally expressing the four ErbB receptors. Down-regulation of cell surface ErbB-1 or ErbB-2 by intracellular expression of specific antibodies has allowed us to delineate the role of these receptors during signaling elicited by: EGF and heparin binding EGF (HB-EGF), ligands of ErbB-1; betacellulin (BTC), a ligand of ErbB-1 and ErbB-4; and neu differentiation factor (NDF), a ligand of ErbB-3 and ErbB-4. Ligand-induced ErbB receptor heterodimerization follows a strict hierarchy and ErbB-2 is the preferred heterodimerization partner of all ErbB proteins. NDF-activated ErbB-3 or ErbB-4 heterodimerize with ErbB-1 only when no ErbB-2 is available. If all ErbB receptors are present, NDF receptors preferentially dimerize with ErbB-2. Furthermore, EGF- and BTC-induced activation of ErbB-3 is impaired in the absence of ErbB-2, suggesting that ErbB-2 has a role in the lateral transmission of signals between other ErbB receptors. Finally, ErbB-1 activated by all EGF-related peptides (EGF, HB-EGF, BTC and NDF) couples to SHC, whereas only ErbB-1 activated by its own ligands associates with and phosphorylates Cbl. These results provide the first biochemical evidence that a given ErbB receptor has distinct signaling properties depending on its dimerization.     

ErbB-1 and ErbB-2 Acquire Distinct Signaling Properties Dependent upon Their Dimerization Partner

The different epidermal growth factor (EGF)-related peptides elicit a diverse array of biological responses as the result of their ability to activate distinct subsets of ErbB receptor dimers, leading to the recruitment of different intracellular signaling networks. To specifically examine dimerization-dependent modulation of receptor signaling, we constructed NIH 3T3 cell lines expressing ErbB-1 and ErbB-2 singly and in pairwise combinations with each other ErbB family member. This model system allowed the comparison of EGF-activated ErbB-1 with ErbB-1 activated by Neu differentiation factor (NDF)-induced heterodimerization with ErbB-4. In both cases, ErbB-1 coupled to the adaptor protein Shc, but only when activated by EGF was it able to interact with Grb2. Compared to the rapid internalization of EGF-activated ErbB-1, NDF-activated ErbB-1 showed delayed internalization characteristics. Furthermore, the p85 subunit of phosphatidylinositol kinase (PI3-K) associated with EGF-activated ErbB-1 in a biphasic manner, whereas association with ErbB-1 transactivated by ErbB-4 was monophasic. The signaling properties of ErbB-2 following heterodimerization with the other ErbB receptors or homodimerization induced by point mutation or monoclonal antibody treatment were also analyzed. ErbB-2 binding to peptides containing the Src homology 2 domain of Grb2 or p85 and the phosphotyrosine binding domain of Shc varied according to the mode of receptor activation. Finally, tryptic phosphopeptide mapping of both ErbB-1 and ErbB-2 revealed that receptor phosphorylation is dependent on the dimerization partner. Differential receptor phosphorylation may, therefore, be the basis for the differences in the signaling properties observed.     

ErbB-4 activation promotes neurite outgrowth in PC12 cells

Neu differentiation factor (NDF; also known as neuregulin) induces a pleiotropic cellular response that is cell type-dependent. NDF and its receptor ErbB-4 are highly expressed in neurons, implying important roles in neuronal cell functions. In the present study we demonstrate that ErbB-4 receptors expressed in PC12 cells mediate NDF-induced signals and neurite outgrowth that are indistinguishable from those mediated by the nerve growth factor-activated Trk receptors. In PC12-ErbB-4 cells but not in PC12 cells, NDF induced an initial weak mitogenic signal and subsequently neurite outgrowth. The NDF-induced differentiation in PC12-ErbB-4 cells was mimicked by the pan-ErbB ligand betacellulin but not by other epidermal growth factor-like ligands. Thus, NDF and betacellulin mediate similar activities through the ErbB-4 receptor. Indeed, only these ligands induced strong phosphorylation of the ErbB-4 receptors. Neurite outgrowth induced by NDF in PC12-ErbB-4 cells was accompanied by sustained activation of mitogen-activated protein kinase (MAPK) and induction of the neural differentiation marker GAP-43. Inhibition of the MAPK kinase MEK or of protein kinase C (PKC) blocked NDF-induced differentiation, whereas elevation of cyclic AMP levels enhanced the response. Taken together, these results indicate that neurite outgrowth induced by ErbB-4 in PC12 cells requires MAPK and PKC signaling networks.     

Single-chain antibody-mediated intracellular retention of ErbB-2 impairs Neu differentiation factor and epidermal growth factor signaling.

ErbB-2 becomes rapidly phosphorylated and activated following treatment of many cell lines with epidermal growth factor (EGF) or Neu differentiation factor (NDF). However, these factors do not directly bind ErbB-2, and its activation is likely to be mediated via transmodulation by other members of the type I/EGF receptor (EGFR)-related family of receptor tyrosine kinases. The precise role of ErbB-2 in the transduction of the signals elicited by EGF and NDF is unclear. We have used a novel approach to study the role of ErbB-2 in signaling through this family of receptors. An ErbB-2-specific single-chain antibody, designed to prevent transit through the endoplasmic reticulum and cell surface localization of ErbB-2, has been expressed in T47D mammary carcinoma cells, which express all four known members of the EGFR family. We show that cell surface expression of ErbB-2 was selectively suppressed in these cells and that the activation of the mitogen-activated protein kinase pathway and p70/p85S6K, induction of c-fos expression, and stimulation of growth by NDF were dramatically impaired. Activation of mitogen-activated protein kinase and p70/p85S6K and induction of c-fos expression by EGF were also significantly reduced. We conclude that in T47D cells, ErbB-2 is a major NDF signal transducer and a potentiator of the EGF signal. Thus, our observations demonstrate that ErbB-2 plays a central role in the type I/EGFR-related family of receptors and that receptor transmodulation represents a crucial step in growth factor signaling.     

ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer.

Overexpression of the erbB-2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB-2-encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB-2 has been reported. We show that in various cells ErbB-2 can form heterodimers with both EGF receptor (ErbB-1) and NDF receptors (ErbB-3 and ErbB-4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB-2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB-2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum-trapped antibody. We report that ErbB-2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB-2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c-Jun kinase (SAPK), by either ligand. Our results imply that ErbB-2 is a pan-ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB-2 in human cancers may be due to its ability to potentiate in trans growth factor signaling.     

The mucin Muc4 potentiates neuregulin signaling by increasing the cell-surface populations of ErbB2 and ErbB3

Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1beta (NRG1beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potentiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression.     

FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion

The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3-induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB-FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.     

The deaf and the dumb: the biology of ErbB-2 and ErbB-3

ErbB-2 (also called HER2/neu) and ErbB-3 are closely related to the epidermal growth factor receptor (EGFR/ErbB-1), but unlike EGFR, ErbB-2 is a ligandless receptor, whereas ErbB-3 lacks tyrosine kinase activity. Hence, both ErbB-2 and ErbB-3 are active only in the context of ErbB heterodimers, and ErbB-2. ErbB-3 heterodimers, which are driven by neuregulin ligands, are the most prevalent and potent complexes. These stringently controlled heterodimers are repeatedly employed throughout embryonic development and dictate the establishment of several cell lineages through mesenchyme-epithelial inductive processes and the interactions of neurons with muscle, glia, and Schwann cells. Likewise, the potent combination of signaling pathways engaged by the heterodimers drives an aggressive phenotype of tumors of secretory epithelia, including breast and lung cancers. This review highlights recent structural insights into the mechanism of ligand-induced heterodimer formation, and concentrates on signaling pathways employed by ErbB-2 and ErbB-3 in normal and in malignant cells.     

Signaling through ShcA is required for transforming growth factor beta- and Neu/ErbB-2-induced breast cancer cell motility and invasion

Cooperation between the Neu/ErbB-2 and transforming growth factor beta (TGF-beta) signaling pathways enhances the invasive and metastatic capabilities of breast cancer cells; however, the underlying mechanisms mediating this synergy have yet to be fully explained. We demonstrate that TGF-beta induces the migration and invasion of mammary tumor explants expressing an activated Neu/ErbB-2 receptor, which requires signaling from autophosphorylation sites located in the C terminus. A systematic analysis of mammary tumor explants expressing Neu/ErbB-2 add-back receptors that couple to distinct signaling molecules has mapped the synergistic effect of TGF-beta-induced motility and invasion to signals emanating from tyrosine residues 1226/1227 and 1253 of Neu/ErbB-2. Given that the ShcA adaptor protein is known to interact with Neu/ErbB-2 through these residues, we investigated the importance of this signaling molecule in TGF-beta-induced cell motility and invasion. The reduction of ShcA expression rendered cells expressing activated Neu/ErbB-2, or add-back receptors signaling specifically through tyrosines 1226/1227 or 1253, unresponsive to TGF-beta-induced motility and invasion. In addition, a dominant-negative form of ShcA, lacking its three known tyrosine phosphorylation sites, completely abrogates the TGF-beta-induced migration and invasion of breast cancer cells expressing activated Neu/ErbB-2. Our results implicate signaling through the ShcA adaptor as a key component in the synergistic interaction between these pathways.     

Epidermal growth factor contains both positive and negative determinants for interaction with ErbB-2/ErbB-3 heterodimers

Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members.     

Extracellular matrix enhances heregulin-dependent BRCA1 phosphorylation and suppresses BRCA1 expression through its C terminus

Germ line mutations in the breast cancer susceptibility gene BRCA1 account for the increased risk of early onset of familial breast cancer, whereas overexpression of the ErbB family of receptor tyrosine kinases has been linked to the development of nonfamilial or sporadic breast cancer. To analyze whether there is a link between these two regulatory molecules, we studied the effects of ErbB-2 activation by heregulin (HRG) on BRCA1 function. It was previously demonstrated that HRG induced the phosphorylation of BRCA1, which was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Since altered interaction between cells and the surrounding extracellular matrix (ECM) is a common feature in a variety of tumors and since ECM modulates intracellular signaling, we hypothesized that ECM may affect the expression and HRG-dependent phosphorylation of BRCA1. Following stimulation by HRG, a strong increase in [(3)H]thymidine incorporation was observed in human T47D breast cancer cells seeded on plastic (PL). When T47D cells were seeded on laminin (LAM) or Matrigel, HRG induced a significantly higher proliferation than it did in cells seeded on PL. T47D cells seeded on poly-L-lysine had an abrogated mitogenic response, indicating the involvement of integrins in this process. HRG treatment induced a transient phosphorylation of BRCA1 that was enhanced in T47D cells grown on LAM. LAM-enhanced BRCA1 phosphorylation was mediated through alpha(6) integrin upon HRG stimulation. Accordingly, T47D cells grown on LAM had the greatest increase in ErbB-2 activation, PI3K activity, and phosphorylation of Akt. A similar pattern of BRCA1 mRNA expression was observed when T47D cells were seeded on PL, LAM, or COL4. There was a significant decrease in the steady state of the BRCA1 mRNA level on both the LAM and COL4 matrices compared to that for cells seeded on PL. In addition, HRG stimulation caused a significant decrease in BRCA1 mRNA expression that was dependent on protein synthesis. Pretreatment with both the calpain inhibitor ALLN (N-acetyl-Leu-Leu-norleucinal) and the proteosome inhibitor lactacystin inhibited the HRG-induced down-regulation of BRCA1 mRNA expression. Likewise, there was a strong decrease in the protein level of BRCA1 in T47D cells 4 h after treatment with HRG compared to its level in control nontreated T47D cells. Pretreatment with the proteosome inhibitors ALLN, lactacystin, and PSI [N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-leucinal] inhibited also the HRG-induced down-regulation of BRCA1 protein in breast cancer cells. Interestingly, BRCA1 mRNA expression in HCC-1937 breast cancer cells, which express C-terminally truncated BRCA1, was not affected by either LAM or CL4. No phosphorylation of BRCA1 from HCC-1937 cells was observed in response to HRG. While Cdk4 phosphorylated wild-type BRCA1 in response to HRG in T47D cells, Cdk4 failed to phosphorylate the truncated form of BRCA1 in HCC-1937 cells. Furthermore, overexpression of wild-type BRCA1 in HCC-1937 cells resulted in the phosphorylation of BRCA1 and decreased BRCA1 expression upon HRG stimulation while overexpression of truncated BRCA1 in T47D cells resulted in a lack of BRCA1 phosphorylation and restoration of BRCA1 expression. These findings suggest that ECM enhances HRG-dependent BRCA1 phosphorylation and that ECM and HRG down-regulate BRCA1 expression in breast cancer cells. Furthermore, ECM suppresses BRCA1 expression through the C terminus of BRCA1.     

Ectopic expression of the ErbB-3 binding protein ebp1 inhibits growth and induces differentiation of human breast cancer cell lines

Ebp1, an ErbB-3 binding protein, translocates from the cytoplasm to the nucleus of human breast cancer cells after treatment with the ErbB-3 ligand, heregulin. The purpose of these studies was to examine the effects of ectopic expression of ebp1 on the biological properties of human ErbB-3-expressing breast carcinoma cell lines. Ectopic expression of ebp1 in ErbB-2, ErbB-3-expressing breast carcinoma cell lines resulted in inhibition of colony formation, a decreased proliferation rate, an accumulation of cells in the G2/M phase of the cell cycle, and suppression of growth in soft agar. Ectopic expression of ebp1 led to a more differentiated phenotype in AU565 breast cancer cells, as evidenced by increased expression of lipid droplets and of the milk protein casein. Basal phosphorylation of extracellular regulated kinases (Erks) 1 and 2, kinases activated by heregulin treatment, was also observed in ebp1 transfectants. The promoter for the intercellular adhesion molecule-1 gene, a heregulin-inducible gene, was constitutively activated in ebp1 transfectants as determined by reporter construct analysis. These data demonstrate that ectopic expression of the ErbB-3 binding protein Ebp1 inhibits proliferation and induces differentiation of ErbB-2, ErbB-3-expressing human breast carcinoma cell lines.     

The Achilles Heel of ErbB-2/HER2: Regulation by the Hsp90 Chaperone Machine and Potential for Pharmacological Intervention

Signal transduction mediated by ErbB/HER receptor tyrosine kinases is crucial for the development and maintenance of epithelial tissues, and aberrant signaling is frequently associated with malignancies of epithelial origin. This review focuses on the roles played by the Hsp90 chaperone machinery in the regulation of signaling through the ErbB/HER network, and discusses potential therapeutic strategies that disrupt chaperone functions. Hsp90 and its associated co-chaperones regulate ErbB signal transduction through multiple mechanisms. The chaperone system controls the stability of the nascent forms of both ErbB-1 (EGF-receptor) and ErbB-2/HER2, while regulation of the mature form is restricted to ErbB-2. Regulation by the Hsp90 complex extends to downstream effectors of ErbB signaling, namely Raf-1, Pdk-1 and Akt/PKB. Disrupting the function of Hsp90 results in the degradation of both the receptors and their effectors, thereby inhibiting tumor cell growth. The importance of an Hsp90-recognition motif located within the kinase domain of ErbB-2 is discussed, as well as a direct role for Hsp90 in regulating tyrosine kinase activity. In light of recent observations, we emphasize the ability of specific tyrosine kinase inhibitors to selectively target ErbB-2 to the chaperone-mediated degradation pathway. ErbB-specific drugs are already used to treat cancers, and clinical trials are underway for additional compounds that intercept ErbB signaling, including drugs that target Hsp90. Hence, the dependence of ErbB-2 upon Hsp90 reveals an Achilles heel, which opens a window of opportunity for combating cancers driven by the ErbB/HER signaling network.     

The achilles heel of ErbB-2/HER2: regulation by the Hsp90 chaperone machine and potential for pharmacological intervention

Signal transduction mediated by ErbB/HER receptor tyrosine kinases is crucial for the development and maintenance of epithelial tissues, and aberrant signaling is frequently associated with malignancies of epithelial origin. This review focuses on the roles played by the Hsp90 chaperone machinery in the regulation of signaling through the ErbB/HER network, and discusses potential therapeutic strategies that disrupt chaperone functions. Hsp90 and its associated cochaperones regulate ErbB signal transduction through multiple mechanisms. The chaperone system controls the stability of the nascent forms of both ErbB-1 (EGF-receptor) and ErbB-2/HER2, while regulation of the mature form is restricted to ErbB-2. Regulation by the Hsp90 complex extends to downstream effectors of ErbB signaling, namely Raf-1, Pdk-1 and Akt/PKB. Disrupting the function of Hsp90 results in the degradation of both the receptors and their effectors, thereby inhibiting tumor cell growth. The importance of an Hsp90-recognition motif located within the kinase domain of ErbB-2 is discussed, as well as a direct role for Hsp90 in regulating tyrosine kinase activity. In light of recent observations, we emphasize the ability of specific tyrosine kinase inhibitors to selectively target ErbB-2 to the chaperone-mediated degradation pathway. ErbB-specific drugs are already used to treat cancers, and clinical trials are underway for additional compounds that intercept ErbB signaling, including drugs that target Hsp90. Hence, the dependence of ErbB-2 upon Hsp90 reveals an Achilles heel, which opens a window of opportunity for combating cancers driven by the ErbB/HER signaling network.