Characterization of the cell membrane-associated products of the Neuregulin 4 gene.

The NRG4 gene is a member of a family of four genes that encode a class of epidermal growth factors. This gene has been reported to express a protein designated here as NRG4A1. We describe here a novel splice variant of the NRG4 gene, NRG4A2, which encodes a C-terminal region containing a predicted type I PDZ-binding peptide. Both NRG4A1 and NRG4A2 were shown to be expressed on the cell surface, as expected by the presence of a predicted transmembrane sequence, and were modified at a single N-linked glycosylation site in the extracellular domain. Significant stabilization of expression of both proteins was seen in the presence of the proteosome inhibitor MG-132 suggesting that they are normally degraded by this system. N-terminal cleavage was inhibited in both isotypes by the broad-spectrum matrix metalloproteinase inhibitor, galardin (GM 6001). A glycosylated, secreted form of NRG4A1 was detected in the cell medium which showed biological activity in two assays, phosphorylation of the HER4 receptor and stimulation of neurite formation in PC-12 cells stably expressing HER4. Transfection and expression of green fluorescent protein-tagged proteins and immunofluorescent staining with specific anti-peptide antibodies showed that NRG4A1 is localized to membrane ruffles, while NRG4A2 has a more punctate membrane distribution.

Proteomic identification of secreted proteins as surrogate markers for signal transduction inhibitor activity.

Epidermal growth factor receptor is a potential target for cancer treatment and new small-molecule tyrosine kinase inhibitor drugs have been designed to inhibit its activity. In this work we identify potential surrogate markers of drug activity using a proteomic analysis. Two-dimensional electrophoresis was optimised to compare expression patterns of proteins secreted from the cancer cell lines A431 and A549 treated with Gefitinib (Iressa) vs untreated or vehicle-only-treated samples. Upregulated or downregulated proteins were detected using Phoretix 2D image analysis software. Several proteins were then identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. In one case, upregulation of Protein Disulphide Isomerase in response to Gefitinib was confirmed by Western blot analysis, and the response was shown to be concentration dependent. The identification of surrogate markers may be of use for the evaluation of new drugs, in preclinical models, in clinical trials and in the therapy of individual patients to give optimal biological drug doses.

Mutations of the epidermal growth factor receptor in non-small cell lung cancer -- search and destroy.

The targeting of the ATP binding pocket of the epidermal growth factor receptor (EGFR) tyrosine kinase, by the small molecule drugs gefitinib and erlotinib, represents a promising new therapeutic strategy in non-small cell lung cancer. However, it is now apparent that only a subset of patients responds to such treatment. Two publications in early 2004 reported the presence of activating mutations in the EGFR tyrosine kinase gene conferring exquisite sensitivity to these drugs. Several publications have since reported prospective data consistent with this finding. This brief review summarises the mutation data from 15 such studies in terms of mutation frequency by clinicopathological features and correlation with response to tyrosine kinase inhibition. A new paradigm for the routine detection of such mutations is needed to facilitate patient selection for treatment and further studies.

Clinical value of epidermal growth factor receptor expression in primary breast cancer.

EGFR expression in primary breast cancer has been extensively investigated for its prognostic and predictive value. However overall there is no consensus on its potential to guide such prognostication. This is largely because of the great heterogeneity in study designs and methods used to assay the EGFR protein. The impetus to standardize such studies is much needed as there are now several tyrosine kinase inhibitors directed against the EGF receptor and phase II trials are showing significant promise.

Consensus statement. Workshop on therapeutic resistance in breast cancer: impact of growth factor signalling pathways and implications for future treatment.

Anti-hormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure anti-oestrogens). However, de novo and acquired resistance comprises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to anti-hormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance anti-tumour actions of existing STI monotherapy, notably including combination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the more complex therapeutic strategies proposed may ultimately be achievable in clinical practice.

The epidermal growth factor receptor family.

The epidermal growth factor receptor family consists of four receptor genes and at least 11 ligands, several of which are produced in different protein forms. They create an interacting system that has the ability to receive and process information that results in multiple outputs. The family has an important role in directing and coordinating many normal processes, including growth and development, normal tissue turnover and wound healing. Its members are also aberrantly activated by overexpression or mutation in many common human tumour types and as such have been the target for anticancer drug development.

Targeted radiosensitisation by pegylated liposome-encapsulated 3', 5'-O-dipalmitoyl 5-iodo-2'-deoxyuridine in a head and neck cancer xenograft model.

5-Iodo-2'-deoxyuridine (IUdR) is an effective radiosensitiser but its clinical development has been limited by toxicity. Prolonged intravenous infusions of IUdR are necessary for optimal tumour uptake but cause dose-limiting myelosuppression. The lack of selective tumour uptake can lead to radiosensitisation of adjacent normal tissues and enhanced local radiation toxicity. Liposomal IUdR delivery offers selective targeting of tumour tissues and avoidance of local and systemic toxicity. In these studies, we report the development of a pegylated liposome containing a lipophilic IUdR derivative (3', 5'-O-dipalmitoyl-5-iodo-2'-deoxyuridine) for use in a head and neck cancer xenograft model. Initial studies confirmed the ability of IUdR to sensitise two head and neck cancer cell lines to single fractions of radiotherapy (SFRT) and this effect was seen to correlate with the thymidine replacement index in KB cells. In vivo delivery of single doses of either unencapsulated IUdR or pegylated liposomal IUdR (PLIUdR) to nude mice bearing KB xenograft tumours did not enhance the effect of SFRT delivered 16 h later. When PLIUdR was delivered by a protracted administration schedule to a dose of 48 mg kg(-1) over 7 days, it enhanced the effect of both 4.5 Gy SFRT and fractionated radiotherapy. PLIUdR was at least as effective as unencapsulated IUdR delivered by multiple intravenous injections or continuous subcutaneous infusion. Immunohistochemistry with a specific anti-IUdR monoclonal antibody confirmed greater levels of tumour staining in tumours from animals treated with PLIUdR compared with those treated with unencapsulated IUdR.

Identification of surrogate markers for determining drug activity using proteomics.

In a high proportion of human carcinomas overexpression of the EGFR (epidermal growth factor receptor), a receptor tyrosine kinase, represents a potential target for cancer treatment. EGFR is induced to dimerize through ligand binding which activates the tyrosine kinase activity of the receptor. This catalyses the transfer of ATP's gamma-phosphate to hydroxyl groups of tyrosine residues on the receptor, creating binding sites that recruit downstream signalling proteins. New drugs, SMTKIs (small-molecule tyrosine kinase inhibitors), have been designed to inhibit the tyrosine kinase activity of the receptor, producing an anti-tumour effect. The development of surrogate markers to determine the drug activity of these new inhibitors would be of great benefit in drug evaluation and in the subsequent management of patient disease. This review describes current treatments of cancer using tyrosine kinase inhibitors and the use of proteomic analysis to identify possible markers of activity of these new drugs.

Type 1 growth factor receptor expression in node positive breast cancer: adverse prognostic significance of c-erbB-4.

BACKGROUND: There is a clear need to define biological markers that will predict the response to treatment in breast cancer, and several recent studies suggest that the expression of type 1 growth factor receptors may prove important in this regard. The type 1 growth factor receptors are a family of transmembrane receptors comprising epidermal growth factor receptor (EGFR), c-erbB-2, c-erbB-3, and c-erbB-4. Both EGFR and c-erbB-2 are associated with poor prognosis in certain tumours. AIMS: There is very little information concerning expression patterns of the full range of type 1 growth factor receptors, especially with respect to c-erbB-3 and c-erbB-4. Therefore, this study was designed to compare the expression of each, and to assess whether expression of any of the factors was related to patient survival in a clinical series. METHODS: Type 1 growth factor receptor expression was investigated by means of immunohistochemistry in a series of node positive patients with breast cancer (n = 66), and statistical analysis was carried out to determine associations between variables and survival analysis for each variable. RESULTS: There were several correlations between variables, and overexpression of EGFR, c-erbB-2, and c-erbB-4 was found to be associated with adverse clinical outcome, although the results were significant only for c-erbB-4 (p = 0.002). CONCLUSION: Although patient numbers are small, this is the first report describing c-erbB-4 as an adverse prognostic marker. These findings are in contrast to previous investigations and may relate to the fact that the patients studied all had advanced stage disease and had undergone similar chemotherapy regimens in the context of a clinical trial.

HER-2 in breast cancer--methods of detection, clinical significance and future prospects for treatment.

The Human Epidermal Growth Factor (HER-2) oncogene encodes a transmembrane tyrosine kinase receptor with extensive homology to the Epidermal Growth Factor Receptor (EGFR) which is the prototypal member of this family of receptor tyrosine kinases. HER-2 gene amplification is found in 20-30% of breast cancers. Various methods such as immunohistochemistry, southern and slot blotting, enzyme immunoassays and fluorescence in situ hybridization have all been employed to evaluate HER-2 gene and protein abnormalities. Of these immunohistochemistry is the most frequently employed but there are valid indications for the other avaliable methods. However, it is prudent that whichever methods employed are standardized, especially those that possess may have a degree of subjectivity in their assesment.

Update on HER-2 as a target for cancer therapy: alternative strategies for targeting the epidermal growth factor system in cancer.

The epidermal growth factor (EGF) family of ligands and receptors interact to influence cell division, differentiation and motility. Much evidence supports their importance in causing and sustaining cell transformation in model systems and in human cancer. The exact mechanism by which this is achieved varies in different tumour types and from case to case. The EGF system is a target for new types of targeted chemotherapy. The choice of strategy will depend on the mechanism involved, however, and several approaches are under development or evaluation in clinical trials. Each will have a different spectrum of side effects and the potential for development of drug resistance.

The Type 1 growth factor receptors and their ligands considered as a complex system.

The Type 1 family of growth factors and their receptors play an important role in normal development, wound healing and in diseases such as cancer. The products of the four receptor genes and the ten genes specifying ligands interact in a complex pattern. Such systems may develop emergent properties which cannot be predicted from a reductionist analysis of the interactions of individual components. New methods of determining these higher level properties are, however, being developed. These include microscopic analysis of live cells expressing fluorescently tagged ligands, receptors and second messengers which can provide positional information on components of the system. Computational simulations of the complex interactions are being developed which can help to predict the properties of the system.

Mutant epidermal growth factor receptor enhances induction of vascular endothelial growth factor by hypoxia and insulin-like growth factor-1 via a PI3 kinase dependent pathway.

Over-expression of truncated epidermal growth factor receptor (EGFR) occurs in a variety of malignancies including glioblastoma multiforme, breast and lung cancer. The truncation deletes an extracellular domain and results in constitutive activation of the receptor. NIH3T3 cells were transfected with full length or truncated human EGFR and differences in growth rates in vivo and in vitro analysed. A growth advantage was seen for cells expressing mutant receptor compared to full length EGFR in vivo only. Administration of an anti-mutant EGFR antibody to mice transiently reduced the growth rates of mutant tumours, confirming that the mutant receptor itself was important in this enhanced tumorigenicity. This showed that stimuli present in vivo and not in vitro may be contributing to growth. We therefore analysed the regulation of the angiogenic factor vascular endothelial growth factor (VEGF). Although levels of secreted VEGF did not differ significantly between wild-type and mutant EGFR cell lines when grown in vitro under normoxic conditions, following exposure to 0.1% hypoxia levels of VEGF produced by mutant cells increased 3.5-6.6 fold compared to 2 or less for full length EGFR cells. The fold induction was influenced by experimental conditions, including cell confluence and percentage of fetal bovine serum, but was consistently higher for mutant cell lines. The increase in VEGF under hypoxic conditions was blocked by the addition of PI3 kinase inhibitors, indicating that the latter pathway is important in the hypoxic stress response. Basal levels were not affected. Addition of insulin-like growth factor-1 also increased levels of VEGF under normoxic conditions in the mutant cells and no further increase was seen when added to cells exposed to 0.1% oxygen, indicating that levels of VEGF were already maximally stimulated. These results show that the mutant EGFR interacts with other growth factors and hypoxia to regulate VEGF via a PI3 kinase pathway, and suggests a specific role for anti-mutant EGFR antibodies and PI3 kinase inhibitors as therapy of this specific tumour target.

Intracellular expression of the truncated extracellular domain of c-erbB-3/HER3.

The ERBB3 gene is expressed as a 6.2- and a 1.4-kb transcript. The former encodes the full-length transmembrane protein and the latter a truncated extracellular fragment consisting of 140 amino acids of the c-erbB-3 protein followed by 43 unique residues. We have examined the expression of the two ERBB3 transcripts by Northern blotting in cancer cell lines and normal human fetal and adult tissues. We expressed the truncated receptor fragment and showed that it was glycosylated, probably with a single N-linked complex sugar chain, and that the protein was a 58-kDa disulphide-linked dimer. We were able to crosslink iodinated neuregulin (NRG)-1beta to the full-length solubilised receptor but not to the truncated dimeric protein. Using Western blot analysis, the truncated protein was shown to be present in cell lysates and, using immunoelectron microscopy, in vesicular structures within cells and associated with the plasma cell membrane.

Expression of erbB-4/HER-4 growth factor receptor isoforms in ovarian cancer.

Immunohistochemical expression of erbB4 protein was identified in 93% (49 of 53) of ovarian cancers using the HFR-1 antibody (targeted to the cytoplasmic domain of the erbB4 receptor) and in 89% (47 of 53) of ovarian cancers using the H4.77.16 antibody (targeted to the extracellular domain). Tumors of serous histology were more likely to express a higher level of erbB4 than endometrioid tumors, and for stage III serous tumors, long-term survival was associated with moderate to high coexpression of erbB4 and erbB2. Within ovarian cancer cell lines, high erbB4 expression was associated with cisplatin resistance. Using reverse transcription-PCR, the presence of multiple isoforms of erbB4 mRNA was identified in both ovarian primary tumors and cell lines. Splice variants in the juxtamembrane (JM-a and JM-d) and cytoplasmic (CT-a and CT-b) regions were identified in mRNA of both cell lines and primary tumors. The use of an anti-erbB4 blocking antibody suggested that erbB4 was not the mediator of the growth stimulatory effects of neuregulin in ovarian cancer cells and indeed could potentially antagonize this effect.

Radiotherapy and cellular signalling.

Developments in cellular and molecular biology in the past decade have increased our understanding of the processes by which cells respond to ionising radiation. Cells use complex protein signalling systems that recognise radiation damage to DNA and plasma membrane lipids. When damage is found, it leads to the activation of various intracellular pathways that modulate the activity of genes controlling ceflular responses such as apoptosis, cell-cycle arrest, or repair. Numerous molecular targets may be activated or inhibited in an attempt to upregulatre or downregulate the radiation response. In this review, we discuss some of the new compounds and techniques for manipulating the cell's response to radiation.

Neuregulin 1-alpha expression in locally advanced breast cancer.

The human Neuregulin 1 (NRG1) gene encodes several alternatively spliced ligands that bind to both c-erbB-3 and c-erbB-4, members of the family of type 1 tyrosine kinase growth factor receptors. Antibodies raised to a synthetic peptide recognize selectively the alpha variant of NRG1. The NRG1-alpha isoforms' expression was studied in 115 locally advanced adenocarcinomas of the breast using immunohistochemistry. Absent or low levels of NRG1-alpha were found to be associated with poorer prognosis compared to tumours that had moderate to high levels of the protein.

Nuclear expression of the c-erbB-4/HER-4 growth factor receptor in invasive breast cancers.

The prevalence and sites of expression of the c-erbB-4 receptor have been determined by immunocytochemical staining in a series of 178 human breast cancers. Most tumors displayed cytoplasmic staining of variable intensity. When compared with adjacent normal tissue, 32 cases (18%) showed lower than normal expression, and 13 (7%) showed greater than normal expression. Nuclear immunoreactivity, confirmed by two different antibodies, was present in 87 cancers (49%) but was found in normal adjacent breast epithelial cells in <5% of cases. There were no significant associations with cytoplasmic or membrane immunoreactivity, but cases showing nuclear expression in >25% of cells were associated with good histological grade, epidermal growth factor receptor expression, c-erbB-3 positivity, cripto, amphiregulin, and transforming growth factor-alpha overexpression.

c-erbB-4 protein expression in human breast cancer.

The Type 1 family of growth factor receptors includes epidermal growth factor receptor (EGFR), c-erbB-2, c-erbB-3 and c-erbB-4. Overexpression of the first two members is associated with poorer prognosis in patients with breast carcinoma. In this study we examined the expression of c-erbB-4 protein using the monoclonal antibody HFR-1. A total of 127 consecutive cases of primary operable invasive breast carcinoma presenting between 1975 and 1977 were studied. All patients were managed by simple mastectomy or conservation surgery with radiotherapy and no adjuvant therapy given. Long-term follow-up was maintained. Routine, formalin-fixed, paraffin-embedded tumour samples were used and sections were stained immunohistochemically using the Duet StreptABC method. Immunoreactivity was classified using a simple semi-quantitative scoring method. Protein expression was generally low but definite positive cytoplasmic, membranous and nuclear reactivity was identified in 58%, 41% and 25% of cases respectively. Expression at all three sites demonstrated significant inverse associations were histological grade. In addition, membrane accentuation correlated inversely with the Nottingham Prognostic Index (NPI), while cytoplasmic reactivity showed a positive association with c-erbB-3 expression. No significant associations were found with disease-free interval or survival. The results of this study demonstrate that higher levels of c-erbB-4 protein expression are associated with a more differentiated histological phenotype in contrast to the other members of the Type 1 family. Larger series with extended follow-up will be required to ascertain definitively the prognostic value of c-erbB-4 expression in breast carcinoma.

Assessment of HER2 status in breast cancer: why, when and how?

Human epidermal growth factor receptor 2 (HER2) is overexpressed, usually as a result of HER2 proto-oncogene amplification, in 20-30% of breast cancers. A HER2-positive status is generally associated with more aggressive disease and a worse prognosis. Furthermore, a positive HER2 status may predict the likelihood of resistance to some conventional therapies, as well as probably being predictive of sensitivity to anthracycline dose intensification. In addition to this prognostic/predictive value, HER2 is a target for specific therapy, with anti-HER2 monoclonal antibody therapy available in the USA. This article reviews the different assays used to determine HER2 status, discussing their relative advantages/disadvantages and the need for their standardisation before integration alongside other pathological indices into the clinical management of breast cancer.