The epidermal growth factor (EGF) receptor family of tyrosine kinases regulates a complex signaling cascade that controls the proliferation, survival, adhesion, migration, and differentiation of cells.[1,2] The dysregulation of EGF receptor signaling by mechanisms such as receptor or ligand overexpression and constitutive activation of receptors can lead to greater cell proliferation and other tumor-promoting activities.[ 3-5] This pathway is therefore tightly regulated in normal cells. The EGF receptor family consists of four distinct receptors: EGFR (ErbB-1), HER2 (HER2/neu, ErbB- 2), HER3 (ErbB-3), and HER4 (ErbB- 4),[2] and their abnormal activation is associated with human cancers of various origins.[6,7] The HER2 gene was cloned in the early 1980s by investigators at Massachusetts Institute of Technology[8,9] and the National Institutes of Health[10] and in Japan.[11] Overexpression of HER2, which has been reported in approximately a third of breast cancers, was found to correlate with tumor resistance to chemotherapy and poor prognosis.[12-15] A meta-analysis that related the expression of HER2 with outcome found that HER2 gene amplification or overexpression of HER2 protein predicted outcome in breast cancer on either univariate or multivariate analysis in 92% of the studies evaluated.[16] Three cellular mechanisms underlie the poor prognosis in patients with HER2-overexpressing tumors.[16-18] First, HER2 overexpression increases the metastatic properties of cancer cells, such as invasion, angiogenesis, and greater survival. Second, HER2 overexpression also confers greater resistance to therapeutic agents (eg, chemotherapy and hormone therapy), which can result in a poor response to treatment. This may correlate with the absence of steroid hormone receptors on HER2-positive cells. Third, HER2 overexpression confers a strong proliferative advantage to tumor cells that are characterized by a high percentage of S-phase cells. In addition, HER2 overexpression has been correlated with larger tumor size and aneuploidy.[ 16,18] This article discusses the molecular mechanisms of the oncogenicity of HER2 overexpression, reviews approaches that use molecular targeting for treating HER2-positive cancers, and describes strategies for optimizing outcomes by using agents that target HER2. Molecular Mechanism: Oncogenicity of HER2 Overexpression All members of the EGF receptor family share a similar structure: they consist of an extracellular ligand-binding domain, a single membrane-spanning region, and an intracellular domain with tyrosine kinase activity. On ligand binding to the extracellular domain, EGF receptors form heterodimers or homodimers, resulting in the activation of intracellular tyrosine kinase and autophosphorylation of specific tyrosine residues.[ 2,19] The phosphotyrosine residues in turn recruit adaptor proteins or enzymes, which initiate signaling cascades to produce a physiologic outcome.[2] Receptor signaling is terminated primarily by endocytosis of the receptor-ligand complex, followed by its recycling to the cell surface or degradation. In normal cells, HER2 does not bind to any known ligand with high affinity, but can signal only by recruiting another activated EGF receptor.[20] The subsequent transactivation and autophosphorylation of HER2 generates intracellular signals that are significantly stronger and of substantially longer duration than signals that emanate from other receptor pairs.[21] There are several molecular reasons for the strength of the signal generated by HER2-containing heterodimers (Table 1). First, HER2 is the preferred heterodimerization partner of all other EGF receptors in normal cells, as well as tumor cells.[22] Overexpression of HER2 in tumor cells may further drive its dimerization by increasing its availability to pair with ligand-activated receptors.[23] HER2 does not bind to any known ligand with high affinity, but on dimerization it increases the affinity of ligands to their receptors by decreasing the rate at which ligands dissociate from the active dimers. In addition, HER2 makes its dimerization partner more promiscuous, allowing it to bind to a broader spectrum of EGF-like ligands.[24] As a result, HER2- containing heterodimers can respond to more ligands with a prolonged and stronger signal. Cancers do not necessarily result from an increased rate of cell proliferation; a disruption of the balance between cell division and cell survival is also a crucial factor. Another basis for the oncogenicity of HER2 stems from the potent activation of both the cell-proliferative Ras-MAPK pathway and the cell-survival pathway that is mediated by PI3K/Akt (Figure 1). Because it does not bind any ligand, HER2 cannot signal directly through these pathways, but it can gain control of the pathways by dimerizing with EGFR and HER3. Epidermal growth factor receptor signaling is terminated by the internalization of cell surface receptors, followed by their degradation. HER2 evades this process of signal attenuation by two mechanisms, resulting in its prolonged signaling.[25] Heterodimers that contain HER2 are internalized more slowly than other heterodimers, resulting in impaired signal attenuation. In addition, HER2- containing heterodimers are not targeted to a degradative pathway; instead, they are recycled to the cell surface. By defective internalization and greater recycling, HER2 heterodimers remain at the cell surface longer, increasing the strength and duration of the intracellular signal. It should be noted that the overexpression of HER2 has been associated with its homodimerization and ligand-independent activation (Figure 1).[26-28] Signaling through the MAPK pathway is also significantly enhanced and prolonged in cells that overexpress HER2 when compared with cells that express low levels.[24] This constitutive activity may play a crucial role in the transformation and proliferation of HER2-positive breast cancer cells. Targeting HER2 in Breast Cancer Because the overexpression of HER2 correlates with the pathogenesis of and prognosis in breast cancer, it is an important therapeutic target. Anti-HER2 therapies (Table 2) reduce the proliferation and survival of tumors that overexpress HER2. Immunologic Therapies
Trastuzumab(Drug information on trastuzumab) (Herceptin), a recombinant humanized monoclonal antibody to the extracellular domain of HER2, is the only anti-HER2 agent that has been approved by the US Food and Drug Administration to treat patients with metastatic breast cancer whose tumors overexpress HER2. Trastuzumab has been shown to have both cytostatic and cytotoxic effects in vitro (Table 3). Trastuzumab disrupts receptor signaling through the downstream proapoptotic PI3K/Akt cell-survival pathway (Figure 2).[29-31] Trastuzumab has also been shown to activate the phosphatase activity of the tumor suppressor PTEN, which reverses the activation of PI3K and Akt.[32] Early studies have shown that trastuzumab induces HER2 internalization and degradation in HER2- overexpressing cells[29,33]; however, recent findings suggest that this may not be true.[34] Cells treated with trastuzumab also undergo growth arrest in the G₁ phase, accompanied by the induction of the cyclin-dependent kinase inhibitor p27.[33,35,36] Trastuzumab has been shown to suppress angiogenesis in vivo by inducing antiangiogenic factors, such as thrombospondin 1, and suppressing proangiogenic factors, such as vascular endothelial growth factor, transforming growth factor, angiopoietin 1, and plasminogen activator inhibitor 1.[37,38] In addition, trastuzumab can block the process of metalloproteinase-mediated HER2 ectodomain shedding, which has been shown to cause constitutive HER2 signaling.[39] By virtue of being an antibody, trastuzumab can harness immune-mediated responses to cause tumor cell toxicity. For example, trastuzumab has been shown to initiate antibodydependent cell-mediated cytotoxicity (ADCC).[40] Trastuzumab has also been shown to potentiate the effects of chemotherapy by multiple mechanisms of action in vitro, as well as in vivo.[41] Pietras and colleagues have shown that treatment with trastuzumab can prevent DNA repair after treatment with DNA-damaging agents.[42] Another molecular explanation for this synergy may be the suppression of the Akt-mediated survival pathway; trastuzumab can therefore induce apoptosis. In fact, a recent trial of trastuzumab in the neoadjuvant setting in primary breast cancers showed that trastuzumab induced apoptosis, confirming that the antibody exerts a cytotoxic effect in vivo.[43] Pertuzumab, another humanized monoclonal antibody to HER2, is currently in phase III trials in patients with breast cancer. In contrast to trastuzumab, pertuzumab binds HER2 near the center of the dimerization arm[44] and can prevent the formation of ligand-induced HER2-containing dimers.[45] As a dimerization inhibitor, pertuzumab diminishes ligandactivated HER2 signaling, including HER2 phosphorylation and activation of MAPK and Akt.[45,46] Pertuzumab would also be expected to recruit effector cells such as macrophages and monocytes to the tumor through the binding of the antibody constant Fc domain to specific receptors on those immune cells. Other antibody-based strategies to attenuate HER2 signaling are in various stages of development. These strategies involve the use of intracellular single-chain Fv antibody fragments as well as armed antibodies. Examples of the latter are toxinlabeled antibodies to HER2[47,48] and antibodies to HER2 labeled with radionuclides such as yttrium-90 and iodine-131.[49,50] Small-Molecule Tyrosine Kinase Inhibitors
The inhibition of tyrosine kinase activity is another strategy for targeting EGF receptor pathways in the treatment of cancer. Small-molecule tyrosine kinase inhibitors (TKIs) have a range of activity, with some specific for a single receptor kinase and others equally active against several receptors.[ 51] Tyrosine kinase inhibitors that are specific for EGFR, eg, gefitinib(Drug information on gefitinib) (Iressa) and erlotinib (Tarceva), have shown only limited efficacy as monotherapies for breast cancer in the preclinical and clinical settings, suggesting that EGFR does not drive tumor growth.[52-54] Dual-kinase inhibitors are a new generation of TKIs that can block signal transduction through both EGFR and HER2 (Table 4). These TKIs inhibit the growth and survival of tumor cells by reducing both MAPK and PI3K signaling (Figure 2).[55,56] Lapatinib, a member of this class of TKIs, has shown promising activity in preclinical and early clinical investigations. In clinical trials lapatinib induced apoptosis and caused growth arrest of tumors that overexpressed HER2 or EGFR.[57] Lapatinib is a reversible inhibitor, and EKB-569 is another dual-kinase inhibitor that irreversibly inhibits the kinase activity of EGFR and HER2; however, the clinical significance of irreversible inhibition has not yet been determined.[51] The potential advantages of dualkinase inhibitors are that they inhibit both ligand-dependent and ligand-independent signaling, they can potentially overcome resistance to trastuzumab in tumors that develop compensatory mechanisms, and they appear to have synergy with chemotherapy. Heat Shock Protein 90 Inhibitors
Another class of small-molecule inhibitors influences EGF signaling by increasing receptor degradation. These inhibitors (eg, geldanamycin) block heat shock protein 90 (HSP90), a chaperone protein that is crucial in maintaining EGF receptors in a signaling- competent form.[58,59] As a consequence, HSP90 inhibitors prevent the stabilization of EGF receptors at the membrane and target these receptors for degradation.[60,61] Geldanamycin, in particular, binds to members of the HSP90 family, blocking the assembly of HSP90 heterocomplexes and destabilizing existing heterocomplexes.[62] As a result, geldanamycin downregulates surface HER2 through greater degradative sorting in endosomes.[34] Treatment with geldanamycin has been shown to result in decreased Akt activity and is correlated with a loss of Akt phosphorylation in breast cancer cells that overexpress HER2.[63] The major drawback of HSP90 inhibitors, however, is their relatively low specificity for EGF receptors; their use can affect the function of many other cellular proteins that require HSP90 for structural stability. Their specificity may be increased by using them in combination with specific anti-EGF receptor TKIs. Other Strategies
Gene therapy strategies for targeting EGF receptor activity aim at blocking the transcription, translation, and maturation of members of EGF receptor transcripts or proteins.[2] Among the agents in development are the adenovirus type 5 early region 1A gene product,[64] triplex-forming oligonucleotides, antisense oligonucleotides, and ribozymes.[65-67] Further experience with these agents may lead to novel strategies that significantly reduce HER2 signaling. Determinants of Clinical Response to Anti-HER2 Agents The clinical benefits of anti-HER2 agents may not be observed in all HER2-positive patients. For example, trastuzumab monotherapy produces an objective response in about a third of patients with HER2-positive disease and clinical benefits in almost half the patients who overexpress HER2.[68] These responses are higher when trastuzumab is used in combination with chemotherapy; objective responses were observed in 50% of patients. Recent studies suggest that there may be a correlation between the decrease in serum concentration of HER2 between 2 and 4 weeks after the start of trastuzumab- based treatment and progression- free survival.[69] The predictive value of serum HER2 levels should be investigated further. It is important to understand the molecular mechanisms that confer resistance to trastuzumab so that patients with disease that will not respond to the therapy are not exposed to its potential adverse effects.[32] Some HER2-positive tumors may have intrinsic resistance to trastuzumab and other anti-HER2 agents, and others may have acquired resistance, ie, they may have developed compensatory mechanisms (discussed in greater detail in the next section). Ongoing research indicates that the intrinsic resistance of tumor cells to trastuzumab may have a number of causes. An in vitro study showed that primary resistance to trastuzumab could stem from a masking of membrane proteins by a membraneassociated mucin.[70] This masking is thought to result in decreased accessibility to and lack of activation of HER2.[70] Evaluation of a HER2- overexpressing breast cancer cell line after exposure to trastuzumab showed an association between downregulation of p27kip1 levels and secondary resistance.[71] Loss or mutation of the tumor suppressor gene PTEN is another important cause of tumor-cell resistance to trastuzumab.[32,72] A retrospective analysis of breast carcinomas found that the responses to trastuzumab-based therapy were significantly poorer in patients with PTEN-deficient tumors than in those with normal PTEN expression.[32] A recent study has elucidated the role of PTEN in increasing sensitivity to HER2 blockers such as trastuzumab. On binding to HER2, trastuzumab stabilizes and activates the PTEN tumor suppressor, thereby downregulating the proapoptotic PI3K/Akt signaling pathway. Thus, PTEN sensitizes tumor cells to trastuzumab, and in the absence of PTEN the antitumor effects of trastuzumab are impaired.[ 32] These findings suggest that drugs that augment PTEN activity may sensitize tumors to trastuzumab. Combinations of HER2 blockers and PI3K inhibitors (once these are commercially available) may have greater efficacy. The mammalian target of rapamycin (mTOR) kinase, which is an important downstream mediator of the PI3K/Akt pathway, is another potential target for overcoming resistance to trastuzumab. The greater proapoptotic activity of PI3K in the absence of PTEN can be attenuated by inhibiting the downstream mediator, mTOR. The addition of mTOR inhibitors, such as sirolimus(Drug information on sirolimus) (rapamycin [Rapamune]) and temsirolimus, may help overcome resistance to trastuzumab in tumors that lack PTEN.[73]