Molecular mechanisms underlying ErbB2/HER2 action in breast cancer

Abstract

Overexpression of ErbB2, a receptor-like tyrosine kinase, is shared by several types of human carcinomas. In breast tumors the extent of overexpression has a prognostic value, thus identifying the oncoprotein as a target for therapeutic strategies. Already, antibodies to ErbB2 are used in combination with chemotherapy in the treatment of metastasizing breast cancer. The mechanisms underlying the oncogenic action of ErbB2 involve a complex network in which ErbB2 acts as a ligand-less signaling subunit of three other receptors that directly bind a large repertoire of stroma-derived growth factors. The major partners of ErbB2 in carcinomas are ErbB1 (also called EGFR) and ErbB3, a kinase-defective receptor whose potent mitogenic action is activated in the context of heterodimeric complexes. Why ErbB2-containing heterodimers are relatively oncopotent is a function of a number of processes. Apparently, these heterodimers evade normal inactivation processes, by decreasing the rate of ligand dissociation, internalizing relatively slowly and avoiding the degradative pathway by returning to the cell surface. On the other hand, the heterodimers strongly recruit survival and mitogenic pathways such as the mitogen-activated protein kinases and the phosphatidylinositol 3-kinase. Hyper-activated signaling through the ErbB-signaling network results in dysregulation of the cell cycle homeostatic machinery, with upregulation of active cyclin-D/CDK complexes. Recent data indicate that cell cycle regulators are also linked to chemoresistance in ErbB2-dependent breast carcinoma. Together with D-type cyclins, it seems that the CDK inhibitor p21Waf1 plays an important role in evasion from apoptosis. These recent findings herald a preliminary understanding of the output layer which connects elevated ErbB-signaling to oncogenesis and chemoresistance.

Introduction

Neu/Her2, also known as ErbB2, was first discovered as a potent oncogenic mutant, when isolated from independent neuroglioblastomas or Schwannomas that developed in carcinogen-treated rats (Schechter et al., 1984). Although this and other ErbB2 mutations are rarely, if at all found in human cancers, wild-type ErbB2 has been often found to be either amplified at the genomic level and/or found to be overexpressed at the protein level. In the majority of cases overexpression correlates with tumor chemo-resistance and poor patient prognosis (reviewed in Hynes and Stern, 1994; Klapper et al., 2000a). This subject has been most extensively addressed in the domain of breast cancer biology, in which ErbB2 is amplified at a 20–30% incidence, and where therapies to subvert ErbB2 expression are now impacting in the clinic.

The first part of this review will address how ErbB2, a member of a complex signaling network, normally functions; an understanding of which is necessary before it can be attempted to elucidate the molecular mechanisms it exploits in cancer. How Her2/ErbB2 overexpression translates into signals that potentiate dysregulated growth, oncogenesis, metastasis and possibly also chemoresistance, particularly in relation to breast cancer, will then be addressed. Not surprisingly, ErbB2 overexpression hyperactivates components of the cell cycle machinery; changes which are also linked to resistance against apoptosis-inducing therapeutic agents. These recent advances towards a clairvoyant understanding of ErbB2 function in cancer, as well as advances in our understanding of the mechanism in which ErbB2-blocking antibodies (Trastuzumab; Herceptin) can in some but not all cases, help facilitate regression of ErbB2-overexpressing tumors will be discussed. This review offers a biochemical and semi-clinical perspective. Reviews directly based on clinical aspects are found elsewhere (e.g. Seminars in Oncology, Volume 26, No. 4. Suppl 12 (1999) Whole Issue; Burris, 2000; DiGiovanna, 1999; Klapper et al., 2000a)). Additionally, a commercially-backed internet site devoted to the use of Herceptin is currently accessible (http://www.prous.com/herceptin).

A mechanistic understanding of ErbB2 function

The ErbB receptor family

ErbB2 is a member of the membrane-spanning type I receptor tyrosine kinase family, comprising four closely related family members, in which ErbB1 (also known as the EGF receptor) was the first to be molecularly cloned (Ullrich et al., 1984). In common with many other growth factor receptors, members of the ErbB family dimerize upon ligand stimulation and transduce their signals by subsequent autophosphorylation catalyzed by the receptor cytoplasmic tyrosine kinase activity, which results in recruitment of an array of downstream signaling cascades (reviewed by Hunter, 2000). The type and amplitude of activated downstream signaling cascades are a co-function of which receptors are expressed by a particular cell, the number of receptors expressed, and the amount and type of ligand that stimulates the cell (Burden and Yarden, 1997; Holland et al., 1998; Schlessinger and Ullrich, 1992).

The heterodimerization model

Although active homodimers can naturally form both for ErbB1 and for ErbB4, ligand-stimulated heterodimer formation is often the norm. In particular, ligand-stimulated heterodimerization is a prerequisite for active signaling for both the ErbB2 and the ErbB3 receptors; ErbB2 binds no known ligand with high affinity, and upon stimulation can only be recruited as a co-receptor with another ErbB member (Klapper et al., 1999). In other words, no known ligand can activate ErbB2 homodimers. Conversely, ErbB3 binds a number of ligands with high affinity, but harbors a defective tyrosine kinase, and thus requires co-recruitment with another ErbB-member to be transactivated (Carraway and Cantley, 1994). A consequence of the ‘heterodimerization model’ is that a particular receptor essentially requires two other signaling components to initiate its activation; namely a co-receptor and a high affinity ligand. Evidence to support this model are described below. The concept of this model is critically important for an understanding of the mechanisms underlying at least early stages of ErbB2-related cancers as it dictates that ErbB2 is not activated in isolation, but rather in concert with at least two other components of its signaling network.

Genetic evidence to support the heterodimerization model

 Developmental genetic studies provide evidence to indirectly support the heterodimerization model. Three examples are described.

  1. 1

    Mouse embryos devoid of functional ErbB2 die at mid-gestation due to a deficiency of ventricular myocardial trabeculation (Lee et al., 1995). A common defect was similarly found in two other mutant mouse strains; one with a targeted disruption of ErbB4 and the other with a disruption of the ErbB-ligand Neuregulin-1 (NRG1) (Gassmann et al., 1995; Meyer and Birchmeier, 1995). Cells expressing NRG1 are juxtaposed to the developing cells of the ventricular myocardium which co-express ErbB2 and ErbB4, demonstrating paracrine cross-talk between these adjacent tissues. Thus there is an essential requirement for these three genes in the same developmental process, consistent with the notion that NRG1-driven ErbB2-ErbB4 heterodimers are essential for heart trabeculae formation.

  2. 2

    NRG1-, ErbB2- and ErbB3-mutant mice all demonstrate a common deficit in precursor Schwann cell development, thus implicating a requirement of NRG1-driven ErbB2-ErbB3 dimers at this developmental checkpoint (Gassmann et al., 1995; Lee et al., 1995; Meyer and Birchmeier, 1995).

  3. 3

    Genetic studies implicate essential roles for ErbB1 in both homo- and heterodimeric complexes. Mice harboring a kinase defective mutant of ErbB1, thus unable to form active ErbB1 homodimers but able to form active heterodimers, exhibit a rather mild phenotype which is very similar to that for mice mutant for the ErbB ligand TGF-alpha (Fowler et al., 1995; Luetteke et al., 1994; Mann et al., 1993; Walker et al., 1998). However ErbB1 knockout mice exhibit extremely severe embryonic abnormalities, implicating an additional requirement for ErbB1 heterodimeric complexes in some developmental processes (Sibilia and Wagner, 1995; Threadgill et al., 1995).

Biochemical evidence for the heterodimerization model

 Cell-lines devoid of endogenous ErbB receptor expression have provided a ‘clean’ experimental system in which to study ErbB function. In these and other cells, stable expression of different ErbB receptors, either singly, or in combinations, has allowed the analysis of ligand-induced ErbB signaling through different homo- and heterodimeric combinations (Alimandi et al., 1997; Cohen et al., 1996a; Pinkas-Kramarski et al., 1996a; Riese et al., 1995; Zhang et al., 1996). Likewise, depletion of ErbB2 expression at the cell surface using either intracellular antibodies (Beerli et al., 1994) or specific ribozymes (Czubayko et al., 1997; Juhl et al., 1997) has allowed the examination of the role of the co-receptor in signaling and tumorigenesis. Importantly, ErbB2 or ErbB3 singly expressed in cells devoid of other ErbBs cannot be activated by ErbB-ligands, even at very high ligand concentrations. However, in the presence of a co-receptor, ErbB2 and ErbB3 promote strong intracellular signaling (Harari et al., 1999; Pinkas-Kramarski et al., 1996a, 1997). Heterodimer formation can be detected by co-immunoprecipitation, trans-phosphorylation and ligand cross-linking analyses. Ligand-induced physical interactions between ErbB proteins was first demonstrated for ErbB1-ErbB2 complexes and later extended for other receptor combinations. In fact, such heterodimeric complexes have been found for every ErbB receptor combination (Tzahar et al., 1996). Thus from a family of four receptors, 10 complexes can be formed: four homodimers and six heterodimers. It is worth mentioning that this complexity evolved relatively late in evolution, as worms and insects express only one ErbB-like receptor (Perrimon and Perkins, 1997). Thus, replacement of the invertebrate linear pathway of ErbB with a mammalian signaling network may represent the need to increase the diversity of this network's output.

Ligands that co-activate ErbB2

Ten distinct mammalian genes, some with numerous alternative splice variants, encode either the ‘EGF receptor ligands’ that bind ErbB1, or the ‘Neuregulins’ that can bind ErbB3 and/or ErbB4, with ErbB2 as a co-receptor. This classification system although valid, is somewhat over-simplistic. To different extents, most ligands have the capacity to bind with high affinity to more than one receptor (Figure 1B). In addition, all ErbB-ligands readily activate ErbB2 in combination with the appropriate high affinity co-receptor. This activation is often of higher affinity and biological potency than complexes lacking ErbB2 (Graus-Porta et al., 1995, 1997; Harari et al., 1999; Karunagaran et al., 1996; Klapper et al., 1999; Riese et al.,1996; Tzahar et al., 1996; Wang et al., 1998). Mammalian ErbB-ligands are mostly membrane spanning, possessing a single transmembrane domain with resultant cytoplasmic and extracellular components. They are structurally divergent, with only a single common trait at the primary structural level; they all encode an extracellular EGF-like domain, which confers ligand-binding capacity (Jacobsen et al., 1996). The characteristic six cysteine residues and conserved spacing of the EGF-like domain predicts the existence of three disulfide bridges, denoted as A, B and C (Figure 1A). The genes NRG1 and NRG2 are unusual in that they harbor two alternate tails of the EGF-like domains (alpha and beta isoforms) which have arisen as a result of exon duplication and differential exon usage (Carraway et al., 1997; Chang et al., 1997; Wen et al., 1994). Currently the role of the transmembrane topology of the precursors of ErbB ligands is not well understood. Whereas experiments performed with pro-TGF-alpha implied that the precursor can interact with a receptor expressed at the surface of a neighboring cell (juxtracrine signaling, reviewed in Massague and Pandiella, 1993), more recent analyses performed in insect and in mammalian cells suggest that transmembrane ligands may be inactive. Proteolytic cleavage of the extracellular domain, resulting in release of the active EGF-like domain seems to be required for ligand activation (Loeb et al., 1998; Schweitzer et al., 1995).

Figure 1
figure1

Multiplicity of ErbB ligands. (a) Alignment of the EGF-like domains of mammalian and viral ligands. ErbB-receptor binding is conferred by the EGF-like domains found for all members of this ligand family, their primary sequences given here. All sequences of the mammalian ligands are human in origin, with the exception of NRG-3 and NRG-4 (mouse). The three pox virus-encoded ligands (bottom) are shown separate from the mammalian ligands. Sequences were aligned using ClustalX software (default parameters). Three predicted disulfide bridges (Cys 1-3, Cys 2-4, Cys 5-6) are labeled as loops A, B and C. Primary references for these sequences are described elsewhere (Harari et al., 1999; Tzahar et al., 1998). Symbols at the bottom represent canonical residues (*), highly conserved amino acids (:), or conserved residues (.). (b) Ligand activation profiles for different ErbB-receptor combinations. Ten possible dimeric complexes of ErbB receptor proteins are represented (columns). The degree of activation by a particular ligand to a receptor combination is indicated by a shaded scale. Activation was based upon physiological mitogenic readouts wherever possible, with exception of NRG-3 where presumed activation was extrapolated from binding data. This composite was derived from numerous sources (Alimandi et al., 1997; Crovello et al., 1998; Graus-Porta et al., 1997; Harari et al., 1999; Jones et al., 1999; Lenferink et al., 1998; Pinkas et al., 1998; Pinkas-Kramarski et al., 1998; Riese et al., 1995, 1996, 1998; Wang et al., 1998; Zhang et al., 1997). (ND) To our knowledge not determined

ErbB2 overexpression potentiates signaling by evading inactivation processes

Ectopic overexpression of ErbB2 to the level observed in breast and other types of tumors enhances tumorigenicity in model systems (Di Fiore et al., 1987; Hudziak et al., 1987). The mechanism underlying the tumorigenic action of an overexpressed ErbB2 may relate to the relatively high basal autophosphorylation activity of this receptor-like tyrosine kinase (Lonardo et al., 1990). Thus, by overexpressing ErbB2 at the cell surface, homodimers may spontaneously form, in analogy to the carcinogen-activated form of the rodent Neu/ErbB2 whose mutation promotes dimerization (Weiner et al., 1989). Alternatively, overexpression of ErbB2 may increase its availability for heterodimer formation when a ligand binds to its direct receptor. Consistent with a heterodimerization model, the transforming action of the rodent mutant depends on a co-expressed ErbB protein (Cohen et al., 1996b). In addition, the transforming ability of ErbB2 is significantly enhanced when co-expressed with either ErbB1 or ErbB3, but in both cases a respective ligand seems essential (Kokai et al., 1989; Wallasch et al., 1995). Thus, an overexpressed ErbB2 may promote carcinogenesis primarily in the context of a ligand-driven heterodimer.

Analyses of the repertoire of signaling molecules that associate with ErbB2 upon autophosphorylation revealed no unique substrate which would explain the transforming action. However, the transforming mutant of ErbB2, which serves as a model system for human cancer, strongly interacts with both the MAPK (Ben-Levy et al., 1994) and the PI3K pathways (Peles et al., 1992). These observations support that cell proliferation and cell survival are activated by ErbB2 through these respective biochemical pathways. Despite the absence of an ErbB2-specific substrate, its signaling ability appears unique in that it is less accessible to normal inactivation processes. For example, signaling through MAPK pathways is significantly prolonged and enhanced in cells overexpressing ErbB2, as opposed to cells whose level of expression is relatively low (Karunagaran et al., 1996). Normally, signaling by growth factor receptors is rapidly inactivated by several mechanisms that safeguard short signals. Dissociation of ligand-receptor complexes, dephosphorylation of the activated receptor, rapid internalization through clathrin-coated pits and degradation of active receptors in lysosomes, ensure rapid termination of signals. With the exception of receptor dephosphorylation, these processes are slowed down when ErbB2 is overexpressed.

  1. 1

    ErbB2 decelerates ligand dissociation rates: Although ErbB2 binds no known ligand with high affinity, when overexpressed it elevates the affinity of EGF (Wada et al., 1990) and neuregulins (Peles et al., 1993; Sliwkowski et al., 1994; Tzahar et al., 1996) to their receptors. The mechanism of increased affinity to multiple ligands can be related to a specific site upon ErbB2, as a class of monoclonal antibodies directed to the extracellular domain of the oncoprotein could accelerate dissociation of several ligands (Klapper et al., 1997). Indeed, overexpression of ErbB2 affects the rate of ligand release from active dimers, but the rate of ligand association with the direct receptor is not affected (Karunagaran et al., 1996). That dimeric receptors bind ligands with higher affinity than monomeric receptors is implied by experiments that made use of mutation-driven dimers of ErbB proteins (Ben-Levy et al., 1992). In conclusion, the stability of receptor dimers, and especially heterodimers with ErbB2, is higher than that of other receptor combinations. This can explain why overexpression of ErbB2 can prolong intracellular signaling, but it leaves open the exact mechanism of ErbB2 involvement. A combination of biochemical, biophysical and mutation mapping analyses have found that ErbB2 can act as a low-affinity and broad-specificity receptor for ErbB-ligands, in contrast to high-affinity binding conferred by other receptors, which has resulted in the inception of a bivalency model of ligand-receptor interaction (Tzahar et al., 1997). In this model, each ErbB-ligand harbors both a high and a low-affinity receptor-binding site. In the case of NRG1, the high-affinity site maps to the N-terminal region of the EGF-like domain, whereas a low-affinity receptor binding site resides on the C-terminal region of the EGF-like domain (Barbacci et al., 1995; Jones et al., 1999; Pinkas-Kramarski et al., 1996a; Tzahar et al., 1996). That ErbB2-containing receptor complexes tend to be the most potent (Graus-Porta et al., 1997; Harari et al., 1999; Jones et al., 1999; Klapper et al., 1999; Riese et al., 1996; Wang et al., 1998), suggests that the bivalency model applies to other ligand-receptor complexes. Therefore, according to this model, one aspect of ErbB2's potent oncogenicity is its preferential binding as a low-affinity receptor to different ErbB-ligands.

  2. 2

    ErbB2 is endowed with a relatively slow rate of endocytosis: Chimeric and wild-type ErbB1 or ErbB2 receptors were employed to demonstrate that the carboxyl-terminus of ErbB2 can reduce 3–4-fold the rate of internalization of ErbB1 (Sorkin et al., 1993). This results in impaired downregulation and degradation of ErbB2, and may explain why heterodimers with ErbB2 initiate relatively long-lived intracellular signals. Likewise, comparative analyses of the rates of internalization of EGF and NRG1 revealed dramatic differences: EGF underwent much more rapid internalization than NRG1, especially when homodimers of ErbB1 were involved (Baulida et al., 1996; Pinkas-Kramarski et al., 1996b). The mechanism responsible for inefficient entrapment of ErbB2 by the clathrin-coated pit is still unknown but it may relate to reduced ability to bind the clathrin-associated protein AP-2.

  3. 3

    ErbB2-containing heterodimers are destined for recycling and their degradation is relatively slow: When ErbB2 is co-expressed with ErbB1, its presence cannot affect the rate of EGF internalization. However, ErbB2 overexpression enhances the rate of recycling of ErbB1 to the cell surface, and at the same time, reduces lysosomal targeting of the EGF-receptor (Lenferink et al., 1998; Worthylake et al., 1999). Because recycling back to the cell surface is primarily a default mechanism, it seems that ErbB2 affects only the rate of delivery to the lysosome. This process is controlled by prior ubiquitination of internalized receptors, a modification that takes place in the early endosome. The mechanism of ubiquitination of ErbB1 has been recently attributed to the activity of c-Cbl, an ortholog of Sli1, the major negative regulator of ErbB signaling in worms (Jongeward et al., 1995). When ErbB1 is activated by EGF, tyrosine 1045 of the carboxy terminus is phosphorylated and recruits c-Cbl. The latter is a ubiquitin ligase that uses a RING finger domain to transfer ubiquitin molecules from an enzyme intermediate to the substrate, ErbB1 (Levkowitz et al., 1998, 1999). By contrast with ErbB1, coupling of ErbB2 to c-Cbl is relatively weak. Although tyrosine 1112 of ErbB2 seems to act as a docking site, ubiquitination and lysosomal targeting of the oncoprotein by c-Cbl are relatively inefficient (Klapper et al., 2000b). In conclusion, lysosomal targeting of ErbB2 is impaired and in the context of a heterodimer ErbB2 can drag ErbB1 to the recycling pathway.

In summary, several distinct mechanisms allow prolonged retention of ErbB2 at the cell surface (Figure 2), thereby extending the duration of signaling by its heterodimeric partners. Especially important is ErbB3, as this kinase-defective receptor is expressed at moderately high levels in most types of human epithelium, and its ectopic co-expression with ErbB2 confers a transformed phenotype (Alimandi et al., 1995; Wallasch et al., 1995). Unlike other receptors, ErbB3 is constitutively recycled to the cell surface and its rate of internalization and degradation are very slow (Baulida et al., 1996; Waterman et al., 1998). The mechanisms underlying mitogenic superiority of the kinase-defective member of the ErbB family were attributed to enhanced interaction with the PI3K pathway and avoidance of the c-Cbl-mediated degradative fate (Waterman et al., 1999).

Figure 2
figure2

Alternative intracellular sorting of ErbB proteins. Binding of a ligand or a monoclonal antibody to ErbB proteins results in accelerated removal from the cell surface through coated regions of the plasma membrane. Invagination of clathrin-coated regions is followed by receptor delivery to endosomes. Subsequently, endocytosed receptors may be targeted to lysosomal degradation. Sorting to the lysosome is mediated by tyrosine phosphorylation and by c-Cbl, a ubiquitin (Ub) ligase that tags incoming receptors with polyubiquitin tracts. As an alternative route, endocytosed receptors may be recycled back to the cell surface, but in either case, the ligands are degraded. Which endocytic route is taken is determined by the identity of the receptor, its dimeric partner and to some extent by the identity of the ligand. Note that homodimers of ErbB1 are destined to lysosomal degradation, whereas ErbB1-ErbB2 heterodimers tend to recycle. Recycling is even more prominent in the case of ErbB3, a receptor whose kinase is defective (marked by a cross) and whose coupling to c-Cbl is inefficient

ErbB2 overexpression in breast cancer

ErbB2

The oncoprotein is overexpresed in 20–30% of breast tumors but particularly so (90%) in comedo forms of Ductal Carcinoma in situ (DCIS), a malignant ductal carcinoma that has not progressed beyond the basement membrane barrier (Barnes et al., 1992; Slamon et al., 1987; van de Vijver et al., 1988). However, even after progression to invasive disease, a correlation with the DCIS subtype and ErbB2 overexpression is maintained (Barsky et al., 1997; Brower et al., 1995). Importantly, high ErbB2 predicts lower disease-free and overall survival in both lymph node negative and particularly in lymph node positive tumors, indicating a functional role of ErbB2 in breast cancer (see Klapper et al., 2000a and references therein). As ligand-dependent stimulation of ErbB2 essentially requires the recruitment of a co-receptor for its activation, another ErbB receptor is thus implicated in ErbB2-dependent cancers. Univariate and co-expression analysis of different ErbBs give no definitive clues as to a single receptor partner that co-activates along with ErbB2.

ErbB1

This receptor seems to be a prime ErbB2 co-receptor candidate; its overexpression or amplification correlates inversely with Estrogen Receptor status, is often expressed in invasive ductal carcinomas and correlates with poor prognosis especially in node negative and perhaps also in node positive patients (Fox et al., 1994; Harris et al., 1992; Nicholson et al., 1991; Torregrosa et al., 1997; VanAgthoven et al., 1995). These phenotypes often overlap with that found for univariate ErbB2 overexpression studies. However, co-expression analyses did not find a significant positive or negative correlation between ErbB1 and ErbB2 overexpression. Cases in which co-overexpression did occur were associated with worse disease free survival prognosis than by either risk factor alone (Torregrosa et al., 1997). These results suggest that ErbB1 plays a similar but not necessarily inter-dependent role in ErbB2-related breast cancers, although their synergy in a subset of tumors does indirectly implicate co-receptor interaction and a more aggressive phenotype.

ErbB3 and ErbB4

Different studies give conflicting reports as to the clinical significance of ErbB3 or ErbB4 expression in breast cancer. If at all, there is a tendency for an observed increase in tumor size, a positive correlation with Estrogen Receptor status and at least for ErbB3, an increased survival prognosis (Bacus et al., 1996a; Gasparini et al., 1994; Knowlden et al., 1998; Lemoine et al., 1992; Quinn et al., 1994; Srinivasan et al., 2000; Travis et al., 1996; Vogt et al., 1998). However, most tumors express either ErbB3 or ErbB4 making statistical assessment of certain tumor subtypes difficult on the basis of differences in receptor expression status alone. It remains an open question if ErbB3 and/or ErbB4 play a significant role in the co-activation of ErbB2 in breast cancers or not. For example, in a pilot-sized study, two-thirds of patients with ErbB2 positive DCIS tumors also exhibited ErbB3 expression (Naidu et al., 1998). As ErbB3 is often expressed in other tumor types, it is difficult to assess if there is a consequential meaning to the co-expression of ErbB2 and ErbB3 or not. It is relevant that initial studies performed with other types of tumors attribute clinical significance to co-expression of ErbB proteins. For example in oral squamous cell cancer co-expression of ErbB2 with ErbB1 or ErbB3 was significant and it critically improved the predicting power (Xia et al., 1999). Future studies on stratified breast tumors may reveal similar relationships.

ErbB ligands

Which of the many ErbB ligands play a critical role in breast cancer is difficult to define as the stroma of the mammary gland is normally enriched for a number of ligands, albeit with different spatial-temporal profiles (Luetteke et al., 1999; Normanno et al., 1994; Herrington, 1997 #4534). Transgenic mouse models overexpressing for example either TGF-alpha or NRG1 under the control of the MMTV promoter demonstrate an accelerated incidence of mammary carcinoma (Krane and Leder, 1996; Sandgren et al., 1990). Tumors usually arise only after a number of months in these mice and are focal in nature, indicating that independent additional genetic events are required for tumorigenesis to occur.

A move to growth factor independence?

Growth factor dependence dictates a requirement for localized ligand stimulation as well as an ErbB co-receptor to maintain ErbB2 signaling for a particular cell. Mammary stroma are enriched for numerous ErbB ligands; some are upregulated in tumors (Ciardiello et al., 1991; de Jong et al., 1998; Lundy et al., 1991; Panico et al., 1996). However, metastatic invasion of tumors to locations such as the lymph nodes, which are presumed poor in ErbB ligand expression may indicate a switch from factor dependence to independence. Two separate mechanisms which can support a factor independent model in ErbB2 or ErbB1 overexpressing tumors are described here. In one model, a naturally but rarely transcribed splice variant of ErbB2 with an in-frame deletion of 16 amino acids in the extracellular domain has been cloned, which is oncogenic in nature and can induce ligand-independent activation of ErbB2 (Siegel et al., 1999). This novel transcript was estimated to be transcribed rarely; from 2–5% of ErbB2 mRNA in total. However, assuming that heterodimers of this gene product with the wild type counterpart result in ligand-independent activation, then potent activation can take place in tumors where ErbB2 is amplified. A second model derived from numerous studies of EGF Receptor (ErbB1) signaling, demonstrate that the EGFR can be trans-activated by a number of heterologous signal transduction pathways. Signaling through different G-protein-coupled-receptors (GPCRs), the growth hormone receptor, interleukin receptors, or voltage-gated calcium channels can all result in the trans-activation of ErbB1 and its downstream components (reviewed by Carpenter, 1999; Zwick et al., 1999). The mechanism of GPCR-mediated trans-activation of ErbB1 was partially solved, where it was found that activation of GPCRs resulted in the cleavage of the ligand HB-EGF from its membrane bound inactive precursor, through the action of a metalloproteinase (Prenzel et al., 1999). This model therefore does not exactly demonstrate ErbB-ligand-independence, but rather the activation of an autocrine loop.

The yin and yang of estrogen receptor and ErbB expression

A negative correlation between ErbB2 and estrogen receptor expression

Numerous studies have shown an intriguing interrelationship between ErbB2 and estrogen receptor (ER) expression and to a lesser extent, progesterone receptor (PR) expression in breast cancer. With exception, there is a strong, highly significant inverse relationship between estrogen receptor expression status and either ErbB2 or ErbB1 overexpression (Battaglia et al., 1988; Roux et al., 1989). Expression of these markers are of clinical significance. ER-negative tumors are more likely to express ErbB1 and/or ErbB2, to be more aggressive and infiltrating in nature and are less often expressed in DCIS. All these trends reversed in patients with ER-positive tumors. A common protocol for patients presented with ER-positive tumors, is to treat them with ‘anti-estrogens’ such as Tamoxifen. Not surprisingly, patients with ErbB2 overexpressing tumors respond poorly to Tamoxifen therapy (Borg et al., 1994; Carlomagno et al., 1996; De Placido et al., 1998; Houston et al., 1999; Newby et al., 1997). However, it seems that ErbB2 signaling can override the tumor-inhibitory effect of anti-estrogens as some studies suggest that when ErbB2 and ER are co-expressed, patients respond poorly to endocrine therapy (Carlomagno et al., 1996; Giai et al., 1994; Houston et al., 1999).

Estrogen represses ErbB2 expression and vice versa

Numerous studies also indicate that a mutually repressive feedback signaling loop exists between ErbB2 expression and that of the ER, probably reflecting the interrelationship of endocrine and paracrine signals important in normal mammary gland development as well as in cancer. Neuregulin administered to several breast cancer cell lines results in repression of ER transcription, although prior to this, a transient upregulation of ER activity has also been observed (Grunt et al., 1995; Matsuda et al., 1993; Mueller et al., 1995; Pietras et al., 1995; Tang et al., 1996). Estrogen administration to breast cancer cell lines results in transcriptional repression of ErbB2. Independent studies found the ErbB2 promoter to be suppressed by either estrogen-induced downregulation or sequestration of the AP-2 or SRC-1 transcription factors respectively (Newman et al., 2000; Perissi et al., 2000).

The Yin and Yang association of ErbB2 to ER mirrors that for ErbB1 and the progesterone receptor (PR), where a dampening feedback loop was uncovered. Progestin administration induced a marked potentiation of EGF/Neuregulin induced signaling in T47D cells, as well as a significant upregulation of ErbB1, ErbB2 and ErbB3 protein levels (Lange et al., 1998). However the progesterone-primed upregulation of ErbB signaling is probably short-lived as PR expression levels were also reported to collapse in cells exposed to EGF for longer periods (Lange et al., 2000). These combined data thus suggest a functional negative interrelationship between steroid signaling and signaling through ErbB2 or ErbB1, and reflect alternate programs activated both during development and in mammary carcinogenesis.

In summary, the inverse relationship between ErbB1 or ErbB2 and steroid receptors may reflect different molecular programs exploited in the genesis of distinct breast cancer subtypes. Nevertheless, even though less common, ER-positive/ErbB2-positive and the reciprocal double-negative breast carcinomas do exist (Sjogren et al., 1998), providing a stern reminder that the negative association between these markers is not absolute.

ErbB2 overexpression hotwires the cell cycle

The cyclin D connection

The D-type cyclins (D1, D2 and D3), which activate their catalytic partners CDK4 and CDK6, are induced by numerous mitogenic stimuli and play a central role in the kick-starting of the cell cycle. Aberrant overexpression of D-type cyclins can reduce or overcome the dependency of mitogenic stimulation for a cell, and thus can play a role in the process of oncogenic transformation (reviewed in Bartkova et al., 1997; Sherr and Roberts, 1999; Weinberg, 1995). Therefore, it may not seem surprising that cyclin D1 plays a pivotal role in breast cancer, being overexpressed at a 40% incidence (Bartkova et al., 1994). Its overexpression is particularly enriched (>75%) in ductal carcinomas in situ (DCIS) (Weinstat-Saslow et al., 1995; Worsley et al., 1997); the majority of which also overexpress ErbB2 (Allred et al., 1992; Mack et al., 1997). Distinct from overexpression, gene amplification of cyclin D1 occurs to a lesser extent, with greater frequency found in both DCIS and invasive lobular carcinoma. Other cyclin genes are rarely, if at all amplified in breast cancer (Bartkova et al., 1994; Courjal et al., 1996; Gillett et al., 1994). These data implicate a generic requirement for cyclin D1 overexpression in different breast cancer subtypes, including those in which ErbB2 plays a role. In line with this notion, one of the two major phenotypes detected in cyclin-D1 knockout mice is a failure of alveolar lobule cells to expand during pregnancy, demonstrating a critical role for cyclin D1 during this period of rapid cell growth (Fantl et al., 1995; Sicinski et al., 1995).

Both estrogen-dependent and ErbB-dependent mitogenic signaling have been shown to channel through the activation of cyclin D1. Using a panel of cell lines transfected with either wild type or oncogenic ErbB2 (NeuT; Val664 to Glu), a dramatic upregulation of cyclin D1 protein expression has been demonstrated (Lee et al., 2000). ErbB2-dependent signaling through cyclins D2 and D3 can also take place (Lane et al., 2000; Neve et al., 2000). This upregulation is at least in part initiated at the transcription level, and involves the SP1 and E2F transcription factors (Lee et al., 2000). ErbB-dependent upregulation of the SP1 transcription factor has been independently reported (Alroy et al., 1999). Cyclin D1 can be upregulated by activated Ras, Raf, MEK and Rac, all downstream targets of ErbB-signaling cascades (Albanese et al., 1995; Aziz et al., 1999; Cheng et al., 1998; Gartel et al., 2000; Gjoerup et al., 1998).

Post-translational stabilization of the normally labile cyclin D1 is also an important process, and can be conferred by its threonine-phosphorylation by PKB/AKT (Diehl et al., 1998). AKT is a major target of ErbB-signaling, specifically found downstream of ErbB3 or ErbB4 and perhaps also ErbB1 signaling complexes, via the activation of PI3 kinase (Elenius et al., 1999; Liu et al., 1999; Moscatello et al., 1998; Prigent and Gullick, 1994; Waterman et al., 1999). AKT activity was down-regulated in cell lines immuno-depleted for ErbB2, thus providing an indirect post-transcriptional link between ErbB expression, PKB activation and cyclin D levels (Lane et al., 2000; Liu et al., 1999; Neve et al., 2000). Taken together, these lines of evidence indicate that D-type cyclins are major down-stream targets of ErbB-dependent signaling, with upregulation possibly taking place both at transcriptional and post-transcriptional levels. A model of ErbB-induced tumorigenicity can thus be construed: ErbB2 amplification results in hyper-activation of a signaling network, which in turn dysregulates the G1/S checkpoint by the formation of high levels of active cyclin D-CDK4/6 complexes.

ErbB2 overexpression confers chemoresistance

Cancer cells overexpressing ErbB2 are often resistant to an array of cytotoxic agents and radiation damage (O'Rourke et al., 1998; Tsai et al., 1993; Yu et al., 1996, 1998b). A mechanistic understanding of how ErbB2 confers chemoresistance remains somewhat elusive. However data to support this claim at the phenomenological level are abundant, and hints as to how ErbB2 overexpressing breast tumors evade not just cell cycle control, but also apoptosis, are beginning to surface.

The first direct evidence supporting that a central mediator of ErbB2's anti-apoptotic machinery may be the CDK inhibitor p21Waf1 (Waf1, Cip1, Sdil) was published 2 years ago (Yu et al., 1998a). This study examined the ErbB2-mediated protective role against paclitaxel (Taxol), normally a highly effective antineoplastic agent which induces apoptosis by interfering with the cell's microtubule machinery (Horwitz, 1992), but of limited therapeutic capacity in ErbB2-expressing tumors. Taxol-treated ErbB2 transfected MDA-MB-435 cells progressed less effectively towards the G2/M phase than control untransfected cells, with a decreased activation of cyclinB-Cdc2 and inhibition of apoptosis (Yu et al., 1998a). In conjunction, the CDK inhibitor p21waf1, was sharply upregulated in ErbB2-overexpressing cells, a phenomenon independently found for a number of cell-lines treated with different ErbB-ligands (Bacus et al., 1996b; Fan et al., 1995, 1997; Fiddes et al., 1998). To test if p21Waf1 was a key mediator of the phenotypic differences observed between ErbB2 high and ErbB2 low expression, the effects of ErbB2 transfected into p21Waf1−/− knockout fibroblasts was examined. Overexpression of ErbB2 in wild-type fibroblasts conferred resistance to taxol-induced cell death, whereas protection against taxol toxicity was abrogated in ErbB2 overexpressing p21−/− cells (Yu et al., 1998a). Thus these data indicate that elevated p21Waf1 levels conferred by ErbB-signaling, elicits changes in the G2/M transition, and can help a cell escape from taxol-mediated cell death.

There are many roads to the upregulation of p21waf1 (reviewed in Gartel and Tyner, 1999). Waf1 is a downstream target of the p53 tumor suppressor, and has been attributed a major role in p53-dependent cell cycle arrest (Deng et al., 1995; el-Deiry et al., 1993). p21Waf1 is also directly upregulated through STAT transcription factors and also indirectly via the Ras-Raf-MAP kinase pathway, both downstream mediators of ErbB signaling (Gartel et al., 2000; Kivinen et al., 1999; Nosaka et al., 1999). Indeed, p21Waf1 was demonstrated to be upregulated in response to NRG1 in different cell types (Bacus et al., 1996b). However, the sharp ErbB2-dependent activation of p21 and resistance to chemotherapy takes place in cells even in the absence of functional p53 (O'Connor et al., 1997; Yu et al., 1998a). Furthermore, depletion of ErbB2 expression results in a subsequent decrease in p21Waf1 levels but not p53 levels (Pietras et al., 1999). These combined data indicate a p53-independent role of p21Waf1 in breast tumors.

Why p21, a CDK inhibitor, may enhance tumorigenicity

p21waf1 was originally identified as a universal CDK inhibitor. It was reported to bind to and inhibit all cyclin/CDK complexes of the cell cycle, with the capacity to induce cell growth arrest (Xiong et al., 1993). Thus it may seem rather perplexing that p21 plays a major role in ErbB2-dependent oncogenicity. However, upon closer inspection, the role of p21 in the cell is far more complex. p21 as well as other CDK ‘inhibitors’ can bind with different stoichiometric ratios to higher order cyclin-CDK complexes. Lower order complexes with a 1 : 1 : 1 subunit ratio of cyclin : CDK : p21Waf1 are thought to confer an active state, whereas additionally bound p21 subunits can at least in some cases, elicit an inhibitory effect (Zhang et al., 1994). Low concentrations of p21waf1 and another CDK inhibitor, p27Kip1, were even demonstrated to enhance the kinetics of assembly of the CDK4/cyclin D complex by more than an order of magnitude over complexes that lacked these ‘inhibitors’. In fact, in primary fibroblasts lacking both p21Waf1 and p27Kip1, cyclin D-CDK complexes failed to form at all (Cheng et al., 1999; LaBaer et al., 1997). Thus members of the p21 CDK family more correctly play activating or inhibitory roles, dependent on their relative expression levels in respect to their corresponding cyclin/CDK cell partners.

Under physiological conditions, it seems that p21Waf1 and p27Kip1, which can act as inhibitors of cyclin E- and A-dependent kinases, also act to positively regulate cyclin D-dependent kinases (Cheng et al., 1999; Polyak et al., 1994; Toyoshima and Hunter, 1994). A major non-catalytic function of cyclin D-dependent kinases is to bind p21 and p27, thus releasing their repressive constraint of these other components of the cell cycle machinery (Geng et al., 1999; Planas-Silva and Weinberg, 1997). Therefore, ErbB2 overexpression in breast cancer cells results in a striking upregulation of cyclin D1-CDK activity, an activity which is not necessarily perturbed by upregulated p21waf1 levels. Interestingly, the literature presents conflicting reports that Neuregulins (NRGs) can induce either proliferation, differentiation or apoptosis in different breast cancer cell-lines (Aguilar et al., 1999; Daly et al., 1997; Guerra-Vladusic et al., 1999; Weinstein et al., 1998). We propose a model in which the fate of a cell in response to ErbB-ligand stimulation is a co-function of the amplitude of ErbB-activation, p53 status and the cyclin D-p21/p27 ratio, where the switch from NRG-dependent differentiation or apoptosis to proliferation is conferred by hyper-activation of the ErbB signaling network (Figure 3).

Figure 3
figure3

Apoptosis, differentiation or proliferation induced by the ErbB-signaling network. A model is proposed in which three key factors contribute towards the phenotypic response to ErbB-signaling: Amplitude of ErbB-signaling as well as levels of both D-type cyclins and p21Waf1. (a) With limiting ligand concentrations or physiological levels of cell surface receptors, signaling through the ErbB network can induce apoptosis, differentiation or cell division, in a delicate balance dictated by cyclin D activation and levels of the cell cycle inhibitor p21Waf1, as well as p53. Other CDK inhibitors, such as p27Kip1 also play a role in the differentiation process (not shown). (b) Amplification of ErbB2 results in hyper-activated signaling of the ErbB signaling network which in turn sharply upregulates cyclin D1 and p21Waf1, resulting in formation of active higher order cyclin D-CDK complexes and subsequent release from the G1/S checkpoint. Some evidence even indicates a pro-survival activity conferred by p21Waf1 overexpression, although the mechanism in which this takes place in the context of breast carcinoma has not been elucidated

Cancer-inhibitory anti-ErbB2 antibodies

The cancer-inhibitory potential of antibodies to ErbB2 was realized as far back as 1984, when mice bearing tumors with an active form of the rodent ErbB2 were treated with specific antibodies (Drebin et al., 1984). However, only within the last 4 years has this realization yielded a method to treat cancer patients (Baselga et al., 1996). Although antibodies conjugated to toxins, radionucleotides and prodrugs show promising results in animal models, it is important to note that even naked forms of antibodies to ErbB2 are active in arresting tumorigenic growth of ErbB2-overexpressing cells. Relevant to the subject of this review is the exact mechanism by which anti-ErbB2 antibodies inhibit cell growth. Experiments performed on various systems suggest that these antibodies can induce differentiation of rapidly dividing breast cancer cells to growth-arrested milk-producing cells (Bacus et al., 1992). Likewise, it has been reported that anti-ErbB2 antibodies down-regulate expression of angiogenic growth factors in tumor cells, both in vitro and in animal models (Petit et al., 1997). Furthermore, ErbB2-mediated resistance to the cytotoxic effects of the tumor necrosis factor can be significantly reduced by monoclonal antibodies to ErbB2 (Hudziak et al., 1988).

Mechanistically, the antibodies appear to act by removing ErbB2 from the cell surface. It has previously been shown that the oncogenic activity of ErbB2 necessitates its localization at the plasma membrane (Flanagan and Leder, 1988). In line with blocking ErbB2 action by preventing its oncogenic interaction with other surface-localized ErbB proteins, the tumor-inhibitory potential of specific antibodies (Hurwitz et al., 1995) or certain combinations of anti-ErbB2 antibodies (Kasprzyk et al., 1992) correlates with the efficiency of antibody-induced down-regulation of ErbB2. Consistent with this notion is the observation that antibody bivalence is essential for inhibitory activity. However, effects mediated by the Fc portion of these antibodies also contribute to their tumor-arresting activity (Clynes et al., 2000). Because all bivalent antibodies to ErbB2 share partial agonistic activity, their ability to down-regulate surface ErbB2 may relate to kinase activation. Indeed, recent evidence suggests that autophosphorylation of ErbB2 and subsequent recruitment of c-Cbl are necessary for antibody-induced down-regulation of ErbB2 (Klapper et al., 2000b).

As a consequence of down-regulation, antibodies to ErbB2 elevate p27Kip1 and the Rb-related protein p130, resulting in a reduction in the number of cells in the S phase of the cell cycle (Sliwkowski et al., 1999). In independent studies, ErbB2-inhibitory antibodies curtailed the levels of p21Waf1 in cells overexpressing ErbB2, although levels of cyclin D1 remained relatively unchanged (Pietras et al., 1998, 1999; Ye et al., 1999). When examining all known cyclin D members, it was found that cyclins D2 and D3 decreased dramatically (Lane et al., 2000; Neve et al., 2000). Thus the downstream effects of blocking antibodies to ErbB2 translate to signals that inhibit progression through the cell cycle and can explain their clinical benefit.

Future perspectives

Our current understanding of the biological role of ErbB proteins in normal physiology and in pathological states is due to the remarkable convergence of experimental data. Molecular cloning of the major players, their studies in vitro and subsequent mutagenesis in model animal systems paved the way to the notion that growth factors and their ErbB surface receptors function in the framework of a communication network. Studies in invertebrates not only helped in tracking the evolution of the network, but also provided genuine physiological perspectives and highlighted the backbone of the primordial network.

In-depth understanding is certainly the key for rational design of therapeutic strategies aimed at the ErbB network. Cancer therapy has taken center stage with the development of tumor-inhibitory antibodies to ErbB1 and ErbB2. Along these lines, novel drugs that inhibit tyrosine kinase activity, interfere with ligand binding, or block transcription of specific components of the network, are expected to reach maturation within the next decade. Some of the pharmacological agents may be useful in the treatments of wound, psoriasis and other hyperproliferative diseases in which the ErbB network is involved.

Despite the overwhelming linkage between basic research and clinical applications, serious gaps in our understanding still exist. For example, we still lack the conceptual framework for cell fate determination. This is best exemplified by the heterogeneity of epithelial organs such as the mammary gland. Construction of a hierarchy of cell lineages, analogous to hematopoiesis, will greatly impact upon the diagnostic and prognostic value of ErbB2, its ligands and their relation to the steroid hormone axis. Such understanding is expected to emerge from studies in animal systems, especially inducible gene targeting and knock-in of mutated components of the ErbB network. On the other extreme, we lack a high resolution structural understanding of the interactions between growth factors and their ErbB receptors. Three dimensional resolution of receptor dimers, especially those containing ErbB2, will allow design of specific antagonists. This promising but currently non-existing avenue awaits successful crystallization of ErbB-ligand complexes.

Lastly, the life of an epithelial or any other type of cell, is controlled by more than one signaling network. Resolution of the interaction between ErbB proteins and G-protein coupled receptors, cytokine receptors, cell adhesion molecules and other networks poses a new challenge to basic researchers. How a convergence of networks is integrated in the cytoplasm and impacts transcriptional programs is still a darkened area. Shedding light on the way information is integrated and translates into specific outputs, such as cell cycle progression, is not just a theoretical issue. For example, the question why specific combinations of anti-ErbB2 antibodies and chemotherapeutic drugs are cardiotoxic, may have not remained an enigma if we understood how the ErbB network interacts with the apoptotic pathway. Undoubtedly more questions will arise from the interface between clinical research and basic research, where we will face more complex challenges.

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Acknowledgements

We thank Drs David Givol, Doron Ginzberg and Patrick Humbert for critical comments, and Mr Fred Meadows for proof-reading the manuscript. Due to space limitation, many original and important studies have not been cited directly, but rather through reviews. Our laboratory is supported by grants from the National Cancer Institute of the US National Institute of Health, Genetech Inc., The Israel Science Foundation and the US Department of Defense Breast Cancer Research Program.

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Correspondence to Yosef Yarden.

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Harari, D., Yarden, Y. Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. Oncogene 19, 6102–6114 (2000). https://doi.org/10.1038/sj.onc.1203973

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Keywords

  • carcinoma
  • growth factor
  • signal transduction
  • tyrosine kinase
  • cyclin D1
  • Waf1

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