During the more than 40 years of research carried out in EGF signalling, our understanding has greatly increased but there is clearly still a great deal to learn about its normal functions. Although fascinating in itself, this research has been further stimulated to an increasing degree by the appreciation of the critical importance of EGF signalling in cancer. Indeed, a significant amount of research conducted by pharmaceutical companies is now directed at exploiting this and closely related systems as targets for drug development.
Cancer cells grow at a rate faster than that at which they die; this is in contrast to cells in normal tissues, in which the rate is balanced or slightly in favour of cell death. It was originally hypothesized that cells cycled at their fastest rate unless they were restrained in some way, perhaps by nutritional or environment limitations, but also by specific factors collectively termed chalones. Despite much endeavour these remained elusive, sometimes with the suspicious characteristic of becoming less active as they became more pure. In the early 1960s, however, Rita Levi-Montalcini purified nerve growth factor and Stanley Cohen (a PhD student in her laboratory) purified EGF [4]. This protein was originally termed 'tooth-lid factor', because the assay for its purification consisted of injecting fractions into newborn mice and measuring the time before their incisors erupted and their eyelids opened; this is not an assay commonly used today!
This, and similar data on purified molecules, strongly supported the concept that cells required positive stimuli to grow. Two observations were critical, fusing the field of growth factor research with that of cancer: the discovery that two retroviruses (simian sarcoma virus and avian erythroblastosis virus) contained a growth factor (platelet-derived growth factor) [5,6] and a mutationally activated growth factor receptor (EGF receptor), respectively, as critical oncogenes [7]. Introduction and expression of these proteins by the virus into susceptible animals or animal cells in culture led to cell transformation. Loss of expression or suppression of their activity made the cells less oncogenic, thereby providing the paradigm that these types of molecules may be responsible for the imbalance in growth that is observed in cancer.
In parallel, work was beginning on the analysis of growth factors and their receptors in human tumour specimens. Hendler and Ozanne [8] first showed, by immunocytochemical staining, that the EGF receptor was present at abnormal levels in human lung cancers. The development of antibodies that could detect expression of these receptors and their ligands in paraffin-embedded, formalin-fixed human tissues enabled larger series of cases to be examined [9]. Although being, in my view, far from complete in terms of accuracy, scale and coverage of molecular types, and certainly in terms of understanding of the information contained, some generalizations have been developed from this research.
Three mutually nonexclusive mechanisms lead to overactivity of growth factor receptors (Fig. 1). Receptors may be present in a normal form at a normal level, but be overactive because of unusually high amounts of ligands produced by a variety of mechanisms. First, through an indirect mechanism, mutations in genes such as Ras lead to increased expression of EGF-like ligands, but it appears that this may only augment cell transformation by Ras itself [10]. Other receptors, in particular G-protein-coupled receptors including gonadotrophin-releasing hormone receptor, caused increased activity of the ADAM family of metalloproteinases, which release active ligands from the cell surface by proteolysis [11]. Gene amplification has not reliably been reported as a mechanism for increased ligand expression (which is an interesting observation because amplification often causes receptor over-expression).
The second mechanism that causes over-activity of growth factor receptor signalling is over-expression of a normal growth factor receptor, either due to increased transcription or gene amplification, or both. It is not clear whether this requires the presence of some ligand or is sufficient to increase the amount of active receptor because of the equilibrium between monomer and dimers. This may be somewhat academic, however, because it is hard to conceive of a cell in an environment in which no ligands exist, but it could have relevance to the choice of and efficacy of different approaches to treatment.
Finally, growth factor receptors can be activated by point mutations (such as the Ret gene in multiple endocrine neoplasia-2A, an inherited predisposition to cancer) [12] and sporadically in the c-kit receptor (in gastrointestinal stromal tumours), but thus far there is no reliable evidence for this occurring in the type 1 receptor family. Deletion of various parts of the EGF receptor gene is quite commonly found in brain tumours, however [13]. The most frequent of these, called the type III mutant EGF receptor, involves the deletion of residues 6–273 in the extracellular domain. This prevents ligand binding, but activates the receptor to approximately 10% of the level achieved by saturating ligand concentrations [14]. In this case the mutated gene is also amplified, suggesting that this level of activation does not achieve full transformation. Various reports have suggested that the type III receptor is expressed at very high prevalence in other tumour types, such as breast cancer. However, the EGFR gene is very rarely amplified in this type of disease and is even less frequently rearranged, so the underlying mechanism producing it must differ, possibly involving tumour specific alternative splicing. In our laboratory, however, we have not found evidence for the mutant receptor by polymerase chain reaction analysis in breast cancer cell lines, or by staining with a mutant receptor protein specific antibody in primary, fixed breast cancer. This issue is important to resolve, because it may be a very promising target for treatment [14].
