Epidermal growth factor receptor: Structure-function informing the design of anticancer therapeutics
Introduction
“I am both amazed and encouraged by the unpredictable scientific and clinical consequences of simply wondering what caused precocious eyelid openings in newborn mice.” – Stanley Cohen [1]
The Epidermal Growth Factor Receptor (EGFR) is a trans-membrane protein implicated in a wide range of developmental biology processes; [2], [3], [4] and human cancers including glioblastoma, non-small cell lung cancer (NSCLC), head and neck cancer and colorectal cancer [5], [6], [7], [8], [9], [10], [11], [12]. The EGFR family has four homologous members: [13], [14] EGFR, also known as ERBB1 or HER1, HER2, also known as ERBB2, HER3 or ERBB3, and HER4 or ERBB4. Each family member has an extracellular domain (ECD) with two cysteine-rich regions, a single trans-membrane, or membrane-spanning region, a juxtamembrane cytoplasmic domain, and an intracellular kinase (or pseudokinase) domain with multiple C-terminal tyrosine residues which are phosphorylated on ligand binding and receptor activation (Fig. 1) [13], [15].
This article provides a framework for understanding new structural insights into the function of the EGFR and describes how new discoveries are informing efforts to improve personalised cancer treatment. We describe the structure, function and targeting of the EGFR family, starting with the extracellular components, i.e. the ectodomains (ECD), proceeding to the transmembrane and juxtamembrane components, and finishing with the intracellular domains, including the tyrosine kinase and C-terminal domains. Some therapeutic strategies, based on our knowledge of the EGFR are considered in the context of dual-target monoclonal antibodies, [16] dual-target inhibitors, [17] ligand-targeting [18] and convection enhanced delivery [19]. The use of these agents to avoid the potential development of resistance to cancer treatment is discussed. Conformational change, [20] oligomerisation, [21] and clustering [22] of EGFR are considered as mechanisms involved in ligand-stimulated kinase activation. This update concludes with a discussion of the relevance of the models of ligand activation, EGFR signaling and recycling for the maintenance of the tumourigenic state and the potential of this information to improve outcomes for cancer patients.
Section snippets
EGFR-family
Growth factor signaling is a critical feature of tissue homeostasis. Since the discovery of Epidermal Growth Factor (EGF), [1] the EGF:EGF-receptor (EGF:EGFR) system has been at the forefront of our knowledge on the structure and function of growth factors, cytokines and cell biology. The EGF:EGFR system is regulated at many levels, the EGFR, the release of activated ligands from their precursors, the induction and activation of the enzymes which release the EGF-like ligands, the processes and
New approaches to ectodomain targeting – disrupting oligomerisation
Dimer or higher-level oligomer formation is important for EGFR activation [84]. The conditions for dimerisation of the EGFR have been studied in detail, [84] and it has been understood for some time that point mutations to either Y246 or Y251, two conserved tyrosines on the oligomerisation arm in domain II of the ECD, both abolish dimerisation and interfere with receptor activation [84], [85], [86]. One strategy to inhibit EGFR signaling is to target the dimerisation arm in the β-hairpin loop
Targeting the transmembrane and juxtamembrane domains of EGFR
The 3D-structure of the EGFR transmembrane domain (TMD) was determined by nuclear magnetic resonance (NMR) spectroscopy [114]. The TMD comprises an initial N-terminal 3–10 turn (residues 644–647) [115] followed by an α-helical region (residues 648–669). [115] Mineev and colleagues [116] studied the conformation of a fragment of the EGFR comprising the juxtamembrane domains and the transmembrane domains (i.e. residues 642–690). On either side of the membrane, the juxtamembrane domains (JMDs) at
The tyrosine kinase domain: structure, mutations, and targeting
The TKD (residues 688–979) is often mutated in cancerous tissue, e.g. L858R- and T790M-EGFR substitutions in NSCLC [138], [139], [140]. The structure of the EGFR-TKD, including 43 aa from the carboxyl-terminal tail, has been solved crystallographically, both free and with erlotinib bound [141]. These 3D-structures also reveal a putative intracellular dimerisation motif which is concentrated in the Leu955-Val956-Ile957 segment lying between the TKD and the carboxyl-terminal tail, as well as
EGFR membrane dynamics
Despite progress in our understanding of the structural biology of the EGFR family, our knowledge of the configuration of the EGFR family members on the cell surface is still incomplete. Recent single-molecule studies [174] have assisted in the investigation of membrane receptor oligomerisation: stepwise photobleaching [175], FRET [176], sub-diffraction localisation microscopy [177], and co-tracking [178]. The distribution of aggregation states for a receptor can now be interrogated at the
EGFR hetero-oligomerisation and clustering
On the cell surface all four ERBB family members are capable of forming heterodimers [192], but HER2 appears to be the preferred dimerisation partner [192]. Since HER2 has no ligand [193] and HER3 is an inactive kinase, [142] it is not surprising that only EGFR and HER4 appear to form homodimers/oligomers which contribute to downstream signaling [192], [194], [195], [196]. It is likely that signaling by HER2/3 heterodimers involves the formation of higher-order oligomers [194]. Using luciferase
EGFR signaling
Historically it has been thought that ligand-induced receptor dimerisation is the key component of EGFR signaling [211], however work done on an EGFR orthologue, the Caenorhabditis elegans LET-23 which is constitutively dimeric [212] suggests EGFR may be regulated by ligand induced allosteric changes in pre-existing receptor dimers [212]. Stimulation of the LET-23 receptor with its ligand LIN-3, appears to respond without alteration in oligomerisation status [212]. When mutational analyses were
EGFR endocytosis and recycling
Inactive receptors are internalised spontaneously, but slowly and these unligated receptors are recycled rapidly back to the cell surface [230]. However, upon ligand binding, active receptors travel through the endosomal system where signaling continues and receptors are either recycled back to the cell surface or taken up into proteolytic lysosomes [231], [232], [233]. Several process including the phosphorylation of the β2 subunit of AP-2 [234], [235], receptor ubiquitylation through the E3
EGFR crosstalk
EGFR signaling can lead to activation of other signaling systems and vice versa. Understanding these cross interactions are important for predicting the effects of regulators on tissue biology.
Earlier discussions in this review indicate that the different EGFR family members can interact directly forming multiple signaling systems: in the EGFR:HER2 both kinases are activated when ligands binds to the EGFR, similarly the EGFR:HER4 is a dual specificity receptor kinase. Although the HER2:HER3
Overcoming resistance and new therapeutic strategies based on targeting the EGFR
As discussed earlier, secondary drug resistance in NSCLC can arise from EGFR-TKD mutations e.g. EGFR-T790M during TKI therapy [164], however, there are intrinsic pathway modulations and feedback loops induced by TKIs which can also lead to drug resistance (Fig. 9). One downstream effect of the action of EGFR kinase inhibitors is the reduction of Akt activity and a consequential reduction in Ets-1 activity [275]. Reduction in Ets-1 activity will reduce the expression of the DUSP6 phosphatase a
Conclusions and future directions
We have discussed recent developments in the structure and function of the EGFR family and their ligands. These discoveries are informing improved development of EGFR targeting drugs and even improving cancer treatment. There are new agents which can disrupt the formation of EGFR oligomers, and by combining EGFR targeting agents with complimentary epitopes, profound downregulation of ligand stimulated EGFR activity can be achieved. In the transmembrane and juxtamembrane domains of the EGFR, the
Acknowledgements
This work was funded in part by the National Health and Medical Research Council through Program Grant no. 1092788. The granting agency had no direct input into this article. R.B.L. is a recipient of the Victorian Cancer Agency Mid-Career Research Fellowship (MCRF15017). R.A.M. is a recipient of a Brain Foundation research grant and a Royal Australasian College of Surgeons Research Scholarship.
Declarations of interest
None.
References (287)
Origins of growth factors: NGF and EGF
J. Biol. Chem.
(2008)A comparison of epidermal growth factor receptor levels and other prognostic parameters in non-samll cell lung cancer
Eur. J. Cancer
(1996)- et al.
Combined anti-Galectin-1 and anti-EGFR siRNA-loaded chitosan-lipid nanocapsules decrease temozolomide resistance in glioblastoma: in vivo evaluation
Int. J. Pharm.
(2015) - et al.
Oligomerization–function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy
Biochim. Biophys. Acta (BBA)-Biomembr.
(2015) Architecture and membrane interactions of the EGF receptor
Cell
(2013)- et al.
VMD: visual molecular dynamics
J. Mol. Graph.
(1996) Synergistic interaction of p185c-neu and the EGF receptor leads to transformation of rodent fibroblasts
Cell
(1989)EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization
Mol. Cell
(2003)Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor α
Cell
(2002)- et al.
Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains
Cell
(2002)
Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor
Cell
Epidermal growth factor-induced hydrogen peroxide production is mediated by dual oxidase 1
Free Radic. Biol. Med.
Amphiregulin
Semin. Cell Dev. Biol.
Betacellulin transgenic mice develop urothelial hyperplasia and show sex-dependent reduction in urinary major urinary protein content
Exp. Mol. Pathol.
Neuregulin-1/ErbB4 signaling regulates visual cortical plasticity
Neuron
Characterization of the binding of 125-I-labeled epidermal growth factor to human fibroblasts
J. Biol. Chem.
Human transforming growth factor-α: precursor structure and expression in E. coli
Cell
Structure and function of epigen, the last EGFR ligand
Semin. Cell Dev. Biol.
GPCRs and EGFR–cross-talk of membrane receptors in cancer
Bioorg. Med. Chem. Lett.
Functions of rhomboid family protease RHBDL2 and thrombomodulin in wound healing
J. Investig. Dermatol.
ADAM metalloprotease-released cancer biomarkers
Trends Cancer
ADAM17, shedding, TACE as therapeutic targets
Pharmacol. Res.
Cullin 3 regulates ADAMs-mediated ectodomain shedding of amphiregulin
Biochem. Biophys. Res. Commun.
EGFR ligands and their signaling scissors, ADAMs, as new molecular targets for anticancer treatments
J. Dermatol. Sci.
CR1/CR2 Interactions modulate the functions of the cell surface epidermal growth factor receptor
J. Biol. Chem.
Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity
J. Biol. Chem.
Structural basis for inhibition of the epidermal growth factor receptor by cetuximab
Cancer Cell
Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype
Science
ADAMs: key components in EGFR signalling and development
Nat. Rev. Mol. Cell Biol.
Determination of EGFR signaling output by opposing gradients of BMP and JAK/STAT activity
Curr. Biol.
Monoclonal antibodies to target epidermal growth factor receptor-positive tumors: a new paradigm for cancer therapy
Cancer
Epidermal growth factor-related peptides and their receptors in human malignancies
Clin. Rev. Oncol./Haematol.
EGF, TGF-alpha and EGFR in human colorectal adenocarcinoma
Acta Oncol.
Overexpression of epidermal growth factor receptor and its ligand transforming growth factor alpha is frequent in resectable non-small cell lung cancer but does not predict tumor progression
Clin. Cancer Res.
Evaluation of epidermal growth factor-related growth factors and receptors and of neoangiogenesis in completely resected stage I-IIIA non-small cell lung cancer- amphiregulin and microvessel count are independent prognostic indicators of survival
Clin. Cancer Res.
A comparative study of epidermal growth factor receptor (EGFR) and MDM2 gene amplification and protein immunoreactivity in human glioblastomas
Pol. J. Pathol.
Genes for epidermal growth factor receptor, transforming growth factor, and epidermal growth factor and their expression in human gliomas in vivo
Cancer Res.
A comprehensive pathway map of epidermal growth factor receptor signaling
Mol. Syst. Biol.
The ErbB-2/HER2 oncogenic receptor of adenocarcinomas: from orphan-hood to multiple stromal ligands
Biochim. Biophys. Acta
Untangling the ErbB SignallingNetwork
Nat. Rev.: Mol. Cell Biol.
Dual targeting of EGFR and HER3 with MEHD7945A overcomes acquired resistance to EGFR inhibitors and radiation
Cancer Res.
Large-scale computational screening identifies first in class multitarget inhibitor of EGFR kinase and BRD4
Sci. Rep.
Peptides targeting EGF block the EGF–EGFR interaction
ChemBioChem
Exploring higher-order EGFR oligomerisation and phosphorylation - a combined experimental and theoretical approach
Mol. Biosyst.
Manipulating the lateral diffusion of surface-anchored EGF demonstrates that receptor clustering modulates phosphorylation levels
Integr. Biol.
Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions
EMBO J.
Neu differentiation factor upregulates epidermal migration and integrin epxression in excisional wounds
J. Clin. Investig.
Molecular aspects of mesenchymal- epithelial interactions
Annu. Rev. Cell Biol.
The cellular response to neuregulins in governed by complex interactions of the erbB receptor family
Mol. Cell. Biol.
Human epidermal growth factor: isolation and chemical and biological properties
Proc. Natl. Acad. Sci. USA
Cited by (59)
The universe of galectin-binding partners and their functions in health and disease
2023, Journal of Biological ChemistryAREG upregulates secreted protein acidic and rich in cysteine expression in human granulosa cells
2023, Molecular and Cellular EndocrinologyCitation Excerpt :Given the role of SPARC in progesterone production, it will be interesting to further investigate the pathophysiological role of SPARC in luteal phase deficiency. EGFR family consists of four members: EGFR (ErbB1), ErbB2, ErbB3, and ErbB4 (Mitchell et al., 2018). The expression of four ErbB receptors can be detected in KGN and hGL cells (Cheng et al., 2022; Wang et al., 2012).
Implicative role of epidermal growth factor receptor and its associated signaling partners in the pathogenesis of Alzheimer's disease
2023, Ageing Research ReviewsCitation Excerpt :The transmembrane domain is a 23-amino-acid helical hydrophobic region linked to the extracellular domain's tip by a proline to keep the receptor immobilized on the membrane. The intracellular domain is separated into three parts: the juxta membrane domain, the tyrosine-protein kinase domain, and the C-terminal domain (Mitchell et al., 2018). All EGFR ligands are type 1 transmembrane precursors, later cleaved at the extracellular domain to generate soluble ligands that bind to activate EGFR.
Immune-related adverse events of cancer immunotherapies targeting kinases
2022, Pharmacology and TherapeuticsCarboranes as unique pharmacophores in antitumor medicinal chemistry
2022, Molecular Therapy OncolyticsCitation Excerpt :The results showed that it was feasible to treat low-metastatic prostate cancer with PSMA containing boric acids or carboranes, thus demonstrating the potential role of PSMA in BNCT for the treatment of prostate cancer.119 Epidermal growth factor receptor (EGFR), a member of the transmembrane RTK family, is involved in promoting growth, proliferation, migration, angiogenesis, and chemotherapy resistance in tumors.113,122–125 Antisense oligonucleotides conjugated with boron clusters (B-ASOs) could serve as potential gene expression inhibitors and boron carriers for BNCT,126–129 providing a dual-action therapeutic platform.