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Stem Cells, Vol. 15, No. 1, 1-8, January 1997
© 1997 AlphaMed Press

Concise Reviews

HER-2/neu Signal Transduction in Human Breast and Ovarian Cancer

David M. Reese, Dennis J. Slamon

Division of Hematology/Oncology and Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California, USA

Key Words. HER-2/neu • erbB receptors • Signal transduction • Breast cancer • Ovarian cancer • Heregulin • Epidermal growth factor

Dr. Dennis J. Slamon, University of California at Los Angeles, Department of Medicine, Division of Hematology/Oncology, 11-934 Factor Building, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA.


   Abstract
Top
 Abstract
Introduction
 Receptor Structure
 Receptor Dimerization and...
 Anti-Her-2 Receptor Antibodies
 Conclusions
 References
 
The HER-2/neu proto-oncogene encodes a 185 kDa transmembrane receptor tyrosine kinase with significant sequence homology to other members of the class I receptor tyrosine kinase family. The HER-2/neu gene is amplified and/or overexpressed in 25%-30% of human breast and ovarian cancers, and overexpression of the receptor is associated with poor prognosis. Tyrosine phosphorylation and activation of the HER-2 receptor lead to activation of specific signal transduction pathways in breast and ovarian cancer cells, including the ras/MAP kinase cascade, phosphatidylinositol 3-kinase, and phospholipase C-{gamma}. HER-2/neu signal transduction pathways ultimately converge on the cell nucleus, where the expression of diverse genes is induced after activation of the receptor. A more complete understanding of HER-2/neu signal transduction pathways may allow the development of specific therapeutics for the treatment of those human breast and ovarian cancers containing this alteration.


   Introduction
Top
 Abstract
 Introduction
Receptor Structure
 Receptor Dimerization and...
 Anti-Her-2 Receptor Antibodies
 Conclusions
 References
 
The HER-2/neu proto-oncogene encodes a 185 kDa transmembrane receptor protein with intrinsic tyrosine kinase activity. HER-2/neu was originally identified as a transforming oncogene in chemically induced rat neuroglioblastomas, where a single point mutation in the transmembrane domain of the molecule is sufficient to confer oncogenic activation [1, 2]. In contrast, the receptor is activated in human breast and ovarian cancers through amplification and overexpression of the wild-type gene. This alteration occurs in 25%-30% of cases and is associated with a poor prognosis [3-6]. HER-2/neu may also play a role in the genesis of human gastric, endometrial and salivary gland cancers [7, 8]. Consistent with these clinical observations, overexpression of HER-2/neu in human breast and ovarian cancer cell lines has been shown to increase DNA synthesis and promote cell growth, improve soft agar cloning efficiency and increase tumorigenicity in nude mouse xenograft models [9-11].

The precise mechanism by which HER-2 overexpression transforms cells remains unknown, but presumably involves activation of the signal transduction pathways through which the receptor operates. We review here some of the current concepts regarding HER-2/neu signal transduction as they relate to its role in the pathogenesis of human breast and ovarian malignancies.


   Receptor Structure
Top
 Abstract
 Introduction
 Receptor Structure
Receptor Dimerization and...
 Anti-Her-2 Receptor Antibodies
 Conclusions
 References
 
HER-2/neu is a member of the erbB/epidermal growth factor receptor (EGFR)/class I family of receptor tyrosine kinases (RTKs). All members of this family, which include EGFR, HER-2/neu, HER-3 and HER-4, are single-chain membrane-spanning proteins which have significant sequence homology to one another [12]. The class I RTKs can be distinguished from at least eight other groups of RTKs on the basis of sequence and structural characteristics [13]. The characteristic structural features of class I RTKs include an extracellular ligand-binding domain containing cysteine-rich regions and possible glycosylation sites, an amphipathic transmembrane region, a short intracellular juxtamembrane region, a tyrosine kinase domain and a carboxyl tail which contains critical tyrosine phosphorylation sites [14]. The highest degree of sequence homology (approximately 80% amino acid identity) between HER-2, EGFR and HER-4 lies in the tyrosine kinase domain, suggesting that this region is essential for the signaling function of these molecules [15]. HER-3, by contrast, has substitutions in four important amino acid residues in its tyrosine kinase domain, leading to the suggestion that this receptor may have reduced or absent enzymatic activity [16]. In experimental systems, HER-3 lacks tyrosine kinase activity when expressed alone in insect cells [17] or a pro-B-lymphocyte line [18], although enzymatic activity has been reported when it is overexpressed in mouse fibroblasts or as a chimera with the EGFR extracellular domain [19].

Structural domains other than the tyrosine kinase region may also play an important role in HER-2/neu signal transduction. The cysteine-rich regions of the extracellular portion of the receptor likely undergo significant disulfide bonding, resulting in specific secondary and tertiary structure, which may influence the molecule's interactions with ligand and other receptors [15]. The juxtamembrane region just inside the plasma membrane contains a conserved site (shared with EGFR) for protein kinase C (PKC) phosphorylation, and phosphorylation of PKC results in downregulation of receptor-binding sites and tyrosine kinase activity [20]. The carboxyl tail regions of class I RTKs display the least degree of sequence homology, perhaps allowing for some degree of signaling specificity. Further insights into the relationships between HER-2/neu receptor structure and function will depend on detailed three-dimensional structural data which are not yet available.


   Receptor Dimerization and Tyrosine Phosphorylation
Top
 Abstract
 Introduction
 Receptor Structure
 Receptor Dimerization and...
Anti-Her-2 Receptor Antibodies
 Conclusions
 References
 
The hallmark of class I RTK activation is phosphorylation on specific tyrosine residues lying primarily in the carboxyl terminal portion of the molecule. Traditionally, activation of this class of receptors was thought to require the formation of receptor homodimers, which transphosphorylate one another and subsequently engage proteins involved in the downstream signal transduction process. This model was developed based on studies of the EGFR. In the case of HER-2/neu, however, it has recently become clear that interactions with other members of the class I RTK family are important for receptor activation, and the nature of these heterodimerization events may determine the cellular response to ligand.

Receptor Dimerization
As stated above, the model for class I RTK homodimerization and activation was originally proposed for interactions between the EGFR molecule and its ligands [21]. For EGFR, activation of single receptor molecules is structurally unfavorable, and ligand binding is thought to induce allosteric changes which favor the formation of homodimers [14]. The extracellular domain is sufficient to promote EGFR homodimer formation [22].

Insights into the role of receptor dimerization in HER-2/neu signal transduction first came from studies of the mutated rat neu protein. A single amino acid substitution within the transmembrane region of the molecule leads to constitutive receptor dimerization and increases in basal tyrosine phosphorylation [23, 24]. This correlates with increased kinase enzymatic activity and phosphorylation of several intracellular targets [25]. Consistent with these data, expression of abbreviated transmembrane fragments of p185, along with the oncogenic form of the neu protein, reduced cell proliferation and inhibited transformation, presumably through inhibition of effective receptor dimerization [26].

Based on these observations, it has been suggested that in human cancer cells overexpressing wild-type HER-2/neu, the receptor dimerizes spontaneously due to the large number of molecules present [27]. In support of this hypothesis, human breast and ovarian cancer cells overexpressing HER-2/neu have high levels of basal tyrosine phosphorylation [28]. Direct study of HER-2 receptor dimerization has been hampered until recently, however, by the lack of a specific ligand known to activate the receptor.

In 1992, heregulin (HRG)/neu differentiation factor (NDF) was identified as a candidate ligand for HER-2/neu on the basis of its ability to induce tyrosine phosphorylation of the molecule in human breast cancer cells [29, 30]. HRG, a 45 kDa secreted protein which is similar to but distinct from other members of the EGF ligand family, exists in multiple isoforms and is known variously as NDF, acetylcholine receptor-inducing activity and glial growth factor, depending on its cell type of origin and reported effects on target cells [29-32]. Despite the fact that it was discovered due to its ability to induce HER-2/neu tyrosine phosphorylation, HRG does not bind directly to HER-2/neu alone. Rather, HRG binds specifically to either the HER-3 or HER-4 molecules alone with relatively low affinity [33, 34]. HER-2, however, is capable of dimerizing with HER-3 and these two molecules together reconstitute a high affinity receptor for ligand [35]. Thus it has been suggested that activation of HER-2/neu may occur only via heterodimerization with other members of the class I RTK family [16], and there is accumulating experimental evidence that this is indeed the case.

It has been known for some time that EGF is also capable of inducing the tyrosine phosphorylation of HER-2/neu in cells which express both receptors, presumably through transphosphorylation of HER-2 by an activated EGFR [36, 37]. The EGFR interaction with HER-2/neu may, in fact, be crucial. The expression of a kinase-negative HER-2 mutant is capable of suppressing normal EGFR signaling after EGF stimulation in a dominant negative fashion [38], and single-chain intracellular retention of HER-2/neu in T47D human breast cancer cells (which express all four class I RTKs at moderate levels) markedly impairs signaling induced by EGF and NDF [39].

HER-2/neu can and does interact with the HER-3 and HER-4 molecules. Evidence for this is found in COS-7 cells expressing HER-2 and HER-3, which form a high affinity receptor for HRG, and in which both receptors can be crosslinked to ligand [35]. In NIH 3T3 cells, neither HER-2 nor HER-3 when expressed alone undergo tyrosine phosphorylation in response to HRG, but when expressed together both are phosphorylated and are associated with enhanced transformation [40]. Likewise, HRG induces the tyrosine phosphorylation of HER-4 but not HER-2 when these receptors are expressed singly in T lymphoblasts, whereas HER-2 is phosphorylated when they are coexpressed [41].

On the basis of these observations, a new model describing HER-2 interactions with the other class I RTKs has been proposed (Fig. 1Go) [16]. According to this scheme, HER-2/neu forms functional receptor heterodimers with each of the other class I RTKs in response to the EGF and HRG families of ligands. HER-2 heterodimerization with other class I RTKs may be an obligate event because as yet there is no known specific ligand for the HER-2/neu receptor. In addition, coexpression of different combinations of class I RTKs with HER-2 may enhance the specificity and expand the repertoire of downstream signaling events available in epithelial cells containing these receptors.



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  		Figure 1. Model for HER-2 interactions with other erbB receptors in human breast cancer. Members of the EGF and HRG family of ligands bind to erbB receptors other than HER-2, which interacts with them in receptor heterodimer pairs to initiate downstream signaling events.

 
Tyrosine Phosphorylation
The sine qua non of class I RTK activation is the phosphorylation of discrete tyrosine sites in the carboxyl terminus of the receptor. In fact, signal transduction by class I growth factor receptors absolutely requires tyrosine kinase activity and tyrosine autophosphorylation [14]. In human breast and ovarian cancer cells, overexpression of HER-2/neu increases basal receptor tyrosine phosphorylation to levels greater than any other cellular substrate, and the degree of HER-2 tyrosine phosphorylation generally correlates with effects on cellular transformation in a dose-dependent fashion [42].

Specific tyrosine sites in the carboxyl tail of HER-2/neu have been identified which may be important for HER-2 signal transduction. Experiments with NIH 3T3 cells expressing human HER-2/neu molecules containing single or multiple mutations in selected carboxyl tyrosine residues show that individual amino acid substitutions generally have no effect on the ability of HER-2 to transform these cells, while the presence of multiple mutations inhibited focal growth in monolayers and was associated with a decrease in HER-2 tyrosine phosphorylation [43]. In addition, studies of a human HER-2 protein containing a transmembrane amino acid mutation similar to the lesion found in oncogenic rat neu protein demonstrated that the additional mutation of tyrosine residue Y1248 reduced the transforming ability of this molecule [27]. The homologous tyrosine residue in the rat neu protein is also essential for the transforming capability of this receptor [44].

While there is substantial evidence implicating specific tyrosine sites in the transforming ability of HER-2/neu, some studies have suggested that deletion of these amino acids does not wholly compromise the ability of HER-2 to transform cells or activate downstream signaling molecules. Indeed, HER-2 mutants with substitutions in single intracellular tyrosine sites retain the ability to transform NIH 3T3 cells and tyrosine phosphorylate Shc, an adaptor molecule which links activated RTKs with the ras/MAP kinase intracellular signal transduction pathway (see below) [45, 46]. In addition, it has been observed that the ability of anti-HER-2 monoclonal antibodies to induce receptor tyrosine phosphorylation may not correlate with their effects on cell proliferation [47], implying that events in addition to simple tyrosine phosphorylation, such as specific conformational changes, may be necessary to effectively activate the receptor. Understanding the specific importance of tyrosine phosphorylation will ultimately require the elucidation of the downstream signaling pathways through which HER-2/neu operates, as well as analysis of their activation in cells bearing HER-2 molecules with alterations in specific tyrosine residues.

Intracellular Signaling Mechanisms
The activation of growth factor receptors leads to the phosphorylation and activation of multiple second messengers within normal cells. Subversion of these intracellular signaling pathways is a common occurence in many human malignancies. Many downstream signaling molecules complex with activated RTKs via src homology 2 (SH2) domains, short modules of approximately 100 amino acids which bind with high affinity to phosphotyrosine residues present in specific amino acid sequences [48]. SH2 domains are present in a number of cellular proteins involved in signal transduction, including enzymes, direct regulators of gene transcription, and molecules which function as adaptors for important protein-protein interactions [48]. Many SH2 domain-containing proteins also have src homology 3 (SH3) domains, which contain a binding site for proline-rich sequences believed to be involved in specific protein-protein interactions [49]. A number of substrates for the HER-2/neu tyrosine kinase containing SH2 and SH3 domains have been identified in human breast and ovarian cancer cells. In addition, other diverse intracellular signaling molecules may play a role in HER-2 signal transduction. An overview of putative HER-2/neu signaling pathways is presented in Figure 2Go.



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  		Figure 2. Postulated HER-2 signal transduction pathways. Activation of the HER-2 receptor induces tyrosine phosphorylation on specific tyrosine residues (YP) in the carboxyl tail of the molecule. Downstream signaling proteins with SH2 domains bind to YP residues in the context of specific flanking amino acid sequences and are phosphorylated. Activation of intracellular signaling pathways occurs and culminates in transcription of nuclear genes. For explanation of individual pathways, see text.

 
The ras/MAP Kinase Cascade
Elucidation of the ras-activated intracellular signaling cascade represents a milestone in modern cell biology. This is a critical pathway for a number of signals transduced inside the cell, many of which are growth-regulating signals. Moreover, ras genes, which encode small proteins with intrinsic GTPase activity, are the most frequently mutated oncogenes in human cancers [50], leading to the concept that this pathway is a common target for many transforming and/or carcinogenic agents. Stimulation of the ras pathway ultimately leads to the activation of mitogen-activated protein (MAP) kinase, an enzyme with a number of intracellular targets including nuclear transcription factors [51]. After tyrosine receptors are phosphorylated, the adaptor molecules grb2 and/or Shc bind to the carboxyl tail of the receptor through SH2 domains [52, 53]. The guanine-nucleotide exchange factor, Sos, next interacts with Shc/grb2 via the grb2 SH3 domain, and this complex is brought to the receptor at the cytoplasmic surface of the plasma membrane. Sos subsequently catalyzes the dissociation of GDP from ras, permitting the formation of an activated ras-GTP complex [54]. Ras-GTP then (through an unknown mechanism which may involve other proteins) activates raf-1, a serine-threonine kinase. raf-1 in turn phosphorylates and activates MEK, a specific threonine-tyrosine kinase which activates MAP kinase [55].

There is evidence that activation of the ras/MAP kinase pathway is important in HER-2/neu signal transduction. In T47D human breast cancer cells, which express all four members of the class I RTK family, Shc phosphorylation and MAP kinase activation occur after treatment with NDF. The degree of MAP kinase activation is markedly reduced, however, when HER-2 expression is decreased by single-chain antibody-mediated intracellular retention of the molecule in the endoplasmic reticulum [39]. Shc and grb2 also bind to HER-2 in both NIH 3T3 cells and human breast cancer cells engineered to overexpress the human HER-2/neu receptor, and Sos can be detected in HER-2 immunoprecipitates [45, 56], presumably indicating activation and complexing of the molecules involved in the ras cascade. We have also observed increases in MAP kinase activity after stimulation of HER-2-overexpressing human breast and ovarian cancer cells with HRG [Arboleda, Reese, Slamon, unpublished observations], and this increase is associated with enhanced transcription of the immediate-early response genes c-fos and EGR1 [57].

Activation of the ras pathway may occur in HER-2-expressing cells through protein interactions which do not involve SH2 domains. Recently, a non-SH2 phosphotyrosine-binding (PTB) domain has been described in the Shc molecule [58], and it appears that HER-2 can interact with this domain [59]. Further studies on the binding of HER-2 to Shc and the activation of the ras cascade are required to determine the relative importance of SH2 and PTB domains to Shc-HER-2/neu interactions.

Phospholipase C-{gamma}
The phosphodiesterase phospholipase C-{gamma} (PLC{gamma}) also binds to activated RTKs via SH2 domains [60]. Phosphorylation of PLC{gamma} activates its enzymatic activity, generating the second messengers inositol 1,4,5-triphosphate and diacylglycerol, which are involved in mobilization of intracellular calcium and possibly activation of raf-1 via protein kinase C [60]. In cells overexpressing human HER-2/neu, PLC{gamma} physically interacts with and is phosphorylated by HER-2 [61]. In addition, PLC{gamma} coimmunoprecipitates with the oncogenic rat neu protein in cells expressing this receptor, and activation of the PLC{gamma} enzyme depends on its phosphorylation by an activated neu receptor [62]. These results indicate that HER-2/neu can physically associate with and tyrosine phosphorylate PLC{gamma}. The functional relevance of this interaction, however, remains to be determined.

Phosphatidylinositol 3-Kinase
Phosphatidylinositol 3-kinase (PI3K) is an enzyme which phosphorylates phosphatidylinositol (PI) and related substrates, generating intracellular second messengers which may have diverse effects on target cells [63]. PI3K is composed of two subunits, a 110 kDa catalytic protein and an 85 kDa regulatory subunit which contains SH2 and SH3 domains [64]. This 85 kDa regulatory subunit interacts with and is phosphorylated by activated growth factor receptors, enhancing the enzymatic activity of the 110 kDa catalytic subunit [63, 64].

PI3K is a substrate for the oncogenic rat neu protein, and neu activation increases PI3K lipid kinase activity [65]. In human breast cancer cells, the p85 subunit of PI3K is phosphorylated by HER-2/neu after ligand-stimulated activation of the receptor, and p85 can be coimmunoprecipitated with activated HER-2/neu [66]. It is possible that activation of PI3K by HER-2/neu is actually mediated by HER-3 in a receptor heterodimer complex, since the HER-3 molecule has a large number of binding domains predicted to interact with p85 and may be unique among human class I RTKs in its ability to couple to PI3K [16, 67, 68]. It has been shown that induction of PI3K lipid kinase activity after stimulation of EGFR with EGF requires interaction between EGFR and HER-3 [69], and it is likely that the same is true in the case of HER-2/neu, although there is no current experimental evidence for this.

Nuclear Transcription Factors
Diverse intracellular signaling pathways ultimately converge on the cell nucleus, where the expression of genes which regulate cellular proliferation and differentiation is tightly coordinated. A number of nuclear transcription factors have been identified as targets of HER-2/neu signal transduction pathways, and induction of their expression may represent the most important result of activation of the HER-2 receptor.

The immediate early nuclear transcription genes, including c-fos, c-jun and EGR1, are rapidly upregulated in response to a variety of stimuli during the G0/G1 stage of the cell cycle, implicating them as fundamental components of the mitogenic response [70]. Treatment of HER-2/neu-expressing T47D human breast cancer cells with NDF results in the transient induction of c-fos and c-jun expression, and downregulation of HER-2 expression blocks this response [39]. In MCF-7 breast cancer cells engineered to overexpress human HER-2/neu, expression of c-fos, EGR1 and the early response gene c-myc can also be induced by HRG, as well as some anti-HER-2 monoclonal antibodies [57]. It is likely that induction of c-fos expression occurs through a ras-dependent pathway because in related RTK systems, mutations of various components of the ras/MAP kinase cascade markedly reduce or eliminate c-fos expression in response to serum growth factors [70-72]. Conversely, the induction of c-jun and c-myc expression may occur through ras-independent signaling mechanisms. A specific pathway leading to the activation of Jun kinase, a member of the MAP kinase family, has recently been identified and is activated in response to some serum growth factors [73, 74]. In addition, there is evidence that induction of c-myc expression after treatment of NIH 3T3 cells with EGF requires functional src, a nonreceptor tyrosine kinase which may couple activated RTKs with induction of c-myc expression [75]. The relevance of these pathways to HER-2/neu signal transduction is currently under investigation.


   Anti-Her-2 Receptor Antibodies
Top
 Abstract
 Introduction
 Receptor Structure
 Receptor Dimerization and...
 Anti-Her-2 Receptor Antibodies
Conclusions
 References
 
HER-2/neu signal transduction has also been studied using antibodies directed against the extracellular domain of the receptor. A variety of antibodies have been generated which have antiproliferative effects on cells overexpressing the HER-2/neu receptor. Some of these antibodies may activate HER-2 signal transduction pathways.

The murine monoclonal antibody 4D5 is directed at the extracellular juxtamembrane region of HER-2/neu and has significant antiproliferative effects on breast and ovarian cancer cells which overexpress this receptor [76-79]. Understanding the effects of the 4D5 antibody on HER-2/neu signal transduction is of particular interest because a humanized version of the molecule [80] has shown promise in the treatment of patients with metastatic breast cancer [81, 82]. In vitro, 4D5 induces the tyrosine phosphorylation of HER-2/neu and leads to partial downregulation of HER-2 receptor levels in target cells [76]. In addition, 4D5 induces PI3K and PI4K lipid kinase activity in breast cancer cells stimulated with the antibody [83]. We have observed that both HRG and 4D5 induce the tyrosine phosphorylation of a comparable suite of downstream signaling molecules including Shc, the p85 subunit of PI3K and PLC{gamma} [Arboleda, Reese, Slamon, unpublished observations], raising the question as to how molecules with opposite biologic effects (cell proliferation versus growth inhibition) differ in their activation of signal transduction pathways.

Other antibodies have been produced which interact with the extracellular domain of HER-2/neu [47, 84, 85]. Shawver et al. studied the effects of a panel of monoclonal anti-HER-2 antibodies on cell proliferation and HER-2/neu signal transduction and found that, while the antibodies varied in their ability to inhibit the growth of HER-2/neu-overexpressing cells and to induce tyrosine phosphorylation of the receptor, there was not a universal correlation between ligand-like effects of the antibodies and their effects on cell proliferation [47]. Other antireceptor antibodies may induce differentiation of HER-2-expressing breast epithelial cells [84], although the mechanisms by which these effects are mediated remain unknown. One potential explanation for the diverse effects of ligand and antireceptor antibodies is that bivalent antibody may cause the formation of obligate HER-2/neu homodimers, which may be capable of tyrosine phosphorylating some intracellular targets but may not generate effective downstream signaling comparable to HER-2 heterodimerization with other members of the class I RTK family (see above). Another possibility is that antibodies and ligand effectively activate different sets of intracellular signaling molecules. Elucidating these differences should prove useful in determining which intracellular signaling pathways are crucial for effective signal transduction through the HER-2 receptor, and may aid in the design of new therapeutic agents directed at HER-2/neu or its signaling pathway.


   Conclusions
Top
 Abstract
 Introduction
 Receptor Structure
 Receptor Dimerization and...
 Anti-Her-2 Receptor Antibodies
 Conclusions
References
 
The HER-2/neu oncogene is amplified and/or overexpressed in 25%-30% of human breast and ovarian cancers, and its overexpression portends a poor prognosis for those patients whose tumors contain this alteration. Activation of specific HER-2/neu signal transduction pathways correlates with the ability of HER-2 to transform breast and ovarian epithelial cells, although characterization of the precise routes through which HER-2 signal transduction is mediated remains incomplete. Improved understanding of HER-2/neu signal transduction pathways may lead to the identification of novel therapeutic targets in the treatment of human breast and ovarian cancer.


   Acknowledgments
 
This work was supported in part by grants 1K12 CA01714 (D.M.R.), R01 CA36827 (D.J.S.), the U.S. Army Breast Cancer Research Program DAMD 17-94-J-4118 (D.J.S.), and the Revlon/UCLA Women's Cancer Research Fund.


   References
Top
 Abstract
 Introduction
 Receptor Structure
 Receptor Dimerization and...
 Anti-Her-2 Receptor Antibodies
 Conclusions
 References
 

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accepted for publication August 9, 1996.




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