Robert W. Akita

Identification of Heregulin, a Specific Activator of p185erbB2

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

Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth.

ErbB2 is a ligand-less member of the ErbB receptor family that functions as a coreceptor with EGFR, ErbB3, and ErbB4. Here, we describe an approach to target ErbB2s role as a coreceptor using a monoclonal antibody, 2C4, which sterically hinders ErbB2s recruitment into ErbB ligand complexes. Inhibition of ligand-dependent ErbB2 signaling by 2C4 occurs in both low- and high-ErbB2-expressing systems. Since the ErbB3 receptor contains an inactive tyrosine kinase domain, 2C4 is very effective in blocking heregulin-mediated ErbB3-ErbB2 signaling. We demonstrate that the in vitro and in vivo growth of several breast and prostate tumor models is inhibited by 2C4 treatment.

Ligand-Independent HER2/HER3/PI3K Complex Is Disrupted by Trastuzumab and Is Effectively Inhibited by the PI3K Inhibitor GDC-0941

Herceptin (trastuzumab) is the backbone of HER2-directed breast cancer therapy and benefits patients in both the adjuvant and metastatic settings. Here, we describe a mechanism of action for trastuzumab whereby antibody treatment disrupts ligand-independent HER2/HER3 interactions in HER2-amplified cells. The kinetics of dissociation parallels HER3 dephosphorylation and uncoupling from PI3K activity, leading to downregulation of proximal and distal AKT signaling, and correlates with the antiproliferative effects of trastuzumab. A selective and potent PI3K inhibitor, GDC-0941, is highly efficacious both in combination with trastuzumab and in the treatment of trastuzumab-resistant cells and tumors.

Spatial control of EGF receptor activation by reversible dimerization on living cells

Epidermal growth factor receptor (EGFR) is a type I receptor tyrosine kinase, the deregulation of which has been implicated in a variety of human carcinomas. EGFR signalling is preceded by receptor dimerization, typically thought to result from a ligand-induced conformational change in the ectodomain that exposes a loop (dimerization arm) required for receptor association. Ligand binding may also trigger allosteric changes in the cytoplasmic domain of the receptor that is crucial for signalling. Despite these insights, ensemble-averaging approaches have not determined the precise mechanism of receptor activation in situ. Using quantum-dot-based optical tracking of single molecules combined with a novel time-dependent diffusivity analysis, here we present the dimerization dynamics of individual EGFRs on living cells. Before ligand addition, EGFRs spontaneously formed finite-lifetime dimers kinetically stabilized by their dimerization arms. The dimers were primed both for ligand binding and for signalling, such that after EGF addition they rapidly showed a very slow diffusivity state that correlated with activation. Although the kinetic stability of unliganded dimers was in principle sufficient for EGF-independent activation, ligand binding was still required for signalling. Interestingly, dimers were enriched in the cell periphery in an actin- and receptor-expression-dependent fashion, resulting in a peripheral enhancement of EGF-induced signalling that may enable polarized responses to growth factors.

Binding specificities and affinities of egf domains for ErbB receptors

ErbB receptor activation is a complex process and is dependent upon the type and number of receptors expressed on a given cell. Previous studies with defined combinations of ErbB receptors expressed in mammalian cells have helped elucidate specific biological responses for many of the recognized gene products that serve as ligands for these receptors. However, no study has examined the binding of these ligands in a defined experimental system. To address this issue, the relative binding affinities of the egf domains of eleven ErbB ligands were measured on six ErbB receptor combinations using a soluble receptor‐ligand binding format. The ErbB2/4 heterodimer was shown to bind all ligands tested with moderate to very high affinity. In contrast, ErbB3 showed much more restrictive ligand binding specificity and measurable binding was observed only with heregulin, neuregulin2β, epiregulin and the synthetic heregulin/egf chimera, biregulin. These studies also revealed that ErbB2 preferentially enhances ligand binding to ErbB3 or ErbB4 and to a lesser degree to ErbB1.

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

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

Preclinical studies with Erlotinib (Tarceva)

Erlotinib HCl (Tarceva; Genentech, Inc, South San Francisco, CA) is an orally available, highly selective, reversible inhibitor of epidermal growth factor receptor (HER1/EGFR) tyrosine kinase. Inhibition of tyrosine kinase activity prevents HER1/EGFR phosphorylation, the associated downstream signaling events, and may block tumorigenesis mediated by inappropriate HER1/EGFR signaling. In vitro and in vivo studies show that erlotinib has activity against human colorectal, head and neck, non-small cell lung, and pancreatic tumor cells. Recent preclinical studies suggest that erlotinib may also have activity against tumors that are dependent on HER2 activation for growth and/or survival. Preclinical studies have addressed the feasibility of using erlotinib in combination with various chemotherapeutic agents, radiotherapy, and targeted agents. Combining agents that have different mechanisms of action has the potential to improve efficacy and inhibit the development of resistance. For example, in preclinical studies, combining erlotinib with cisplatin, doxorubicin, gemcitabine, or low-dose paclitaxel has an additive effect on antitumor activity with no increase in toxicity. Preclinical data provide a strong rationale for investigating erlotinib in the clinical setting. However, additional studies are required to gain further insights into the processes that regulate or influence the antitumor activity of erlotinib.

Erlotinib directly inhibits HER2 kinase activation and downstream signaling events in intact cells lacking epidermal growth factor receptor expression.

Erlotinib (Tarceva), is an orally available, reversible inhibitor of epidermal growth factor receptor (EGFR; HER1) that exhibits inhibitory activity on purified HER2 kinase at much higher concentrations. Despite the minimal activity on purified protein in vitro, in vivo studies show that erlotinib inhibits the growth of HER2-driven systems effectively. Several hypotheses have been put forward to explain this discrepancy. In particular, it has been suggested that erlotinib might indirectly suppress the activity of HER2 by blocking the ability of EGFR to transactivate it when the two receptors are part of a heterodimer complex. However, an alternative possibility that has not been adequately addressed is whether the direct inhibitory action of erlotinib on the HER2 kinase might account for the observed biological responses. To distinguish between a direct effect of erlotinib on HER2 kinase in intact cells or an indirect effect of erlotinib on HER2 activity that is mediated through EGFR, we generated cell lines that express either EGFR-H2 chimeric receptor or HER2 and HER3 receptors in an EGFR-negative background. We show that dose-dependent inhibition of HER2 was achieved at the receptor level, on downstream signaling molecules, and more importantly was also translated into inhibition of cell growth. Our findings imply that the inhibitory effect of erlotinib in HER2-expressing cells may in part be mediated through direct interaction with HER2 rather than indirectly through a process that requires the presence of EGFR.

Identification of a region within the ErbB2/HER2 intracellular domain that is necessary for ligand-independent association.

Ligand-independent ErbB2 activation occurs principally by two distinct mechanisms: overexpression and mutation. Overexpression of ErbB2 at the plasma membrane drives receptor self-association in a concentration-dependent manner, which in turn leads to constitutive receptor activation. Subsets of human breast cancers contain a molecular alteration that leads toerbB2 gene amplification and subsequent protein overexpression. Although not recognized to occur in human cancers, mutation can also lead to increased ErbB2 association. A well characterized mutant of the rodent ortholog neu involves substitution of glutamate for valine within the transmembrane domain. In each case, a number of explanations have been proposed to explain the resulting ErbB2 activation. These include stabilization of receptor oligomers, release of negative constraints, and altered receptor conformations. Here we define a short amino acid segment comprising amino acids 966–968 in the intracellular domain that seemingly disrupts receptor-receptor association that is driven either by overexpression or mutation in the transmembrane region. Because of the hydrophobic nature of these amino acids (VVI), we propose that alteration of this segment likely results in a global conformational change in an area that has been proposed previously to be a dimerization motif for ErbB homomeric association.

Structural Requirements for ErbB2 Transactivation

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