New developments in the molecular biology of breast cancer.
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Breast Neoplasms↗
Biomedical subjects
W J Gullick
Publications and source records attributed to W J Gullick.
Absence of abnormalities of the c-erbB-1 and c-erbB-2 proto-oncogenes in human thyroid neoplasia.
The c-erbB-1 and c-erbB-2 proto-oncogenes are frequently activated by gene amplification and overexpression in a variety of human cancers. In an analysis of a large series of benign and malignant thyroid tumours, no abnormalities of structure or expression of either of c-erbB-1 or c-erbB-2 were found. Activation of these oncogenes is not a necessary event in neoplasia of this epithelial system.
DNA, Neoplasm↗
Growth factors and oncogenes in breast cancer.
The growth of normal breast epithelial cells is regulated by a complex interacting system of polypeptide factors and by steroid hormones. The cells respond to these factors through receptors which generate mitogenic and other intracellular signals. These second messengers provoke complex responses which may ultimately result in DNA replication and cell division. A comparison of normal cells and tumour cells, either in culture or from primary tumour biopsies, has revealed differences in growth factor and growth factor receptor expression. Such changes may represent aspects of the process of malignant transformation. In addition some evidence suggests that changes in second messenger systems may also occur. Finally several changes in nuclear oncogenes have been observed in breast cancers. It has been proposed that changes in the nuclear oncogenes, perhaps involving the loss of function of tumour suppressor genes, may allow cells to enter the cell cycle. Changes in growth factors, their receptors or intracellular second messenger systems may stimulate unregulated growth. The combination of these events provide a model for the process of carcinogenesis.
Breast Neoplasms↗
c-erbB-2 protein expression in node negative breast cancer.
We investigated the expression of c-erB-2 protein in two matched groups of breast cancer patients, one with and one without relapse. 37 patients with relapse were compared with 42 patients without recurrence for time of observation, adjuvant treatment, age, menopausal status and estrogen receptor content. Paraffin-embedded sections were stained with the polyclonal antibody 21N, raised against a synthetic peptide from the predicted sequence of the c-erbB-2 protein. The staining of c-erbB-2 was measured on a scale of 0 to 3+. C-erbB-2 staining was negative in 16 (38%) patients in the relapse-free group, and in 8 (22%) of the patients with metastases. Neither disease-free survival (DFS) nor overall survival (OS) were dependent upon the extent of c-erbB-2 expression. An analysis by estrogen receptor (ER) status (i.e. positive or negative) and by c-erbB-2 expression (i.e. positive or negative) revealed that patients with ER-positive primaries and negative c-erbB-2 have the longest disease-free survival (DFS) and overall survival (OS). We conclude that c-erbB-2 expression might be clinically useful only if other prognostic variables (e.g. estrogen receptor content in the tumor) are also considered.
Adult↗
A sequence motif in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization.
A mechanism by which ligand binding to the extracellular domain of a growth factor receptor causes activation of its cytoplasmic tyrosine kinase domain is that binding promotes receptor dimerization. Recently we proposed a model in which dimerization of the transmembrane alpha-helices in one member of this family, rat neu, is mediated by the presence of three specific residues. This paper shows that a similar sequence motif is observed in 18 of the 20 transmembrane alpha-helices of the tyrosine kinase family of growth factor receptors. The motif encompasses a five residue segment in which position 0 (P0) requires a small side chain (Gly, Ala, Ser, Thr or Pro), P3 an aliphatic side chain (Ala, Val, Leu or Ile) and P4 only the smallest side chains (Gly or Ala). In addition other features of the transmembrane sequences are reported. It is concluded that the dimerization of transmembrane alpha-helices may be a general mechanism of tyrosine kinase activation in this family of growth factor receptors.
Amino Acid Sequence↗
Absence of activating transmembrane mutations in the c-erbB-2 proto-oncogene in human breast cancer.
The rat neu proto-oncogene, which is a putative growth factor receptor closely related to the epidermal growth factor receptor, can be activated in vivo by a single point mutation in the sequence encoding its transmembrane region. The human homologue of neu, c-erbB-2, can be activated in vitro to an oncogenic form by point mutations in the same relative position in the gene. We have sought the presence of such activating mutations in a series of 100 cases of human breast cancer by polymerase chain reaction amplification and sequence-specific oligonucleotide hybridization, and also by a designed restriction fragment length polymorphism strategy in cases with Southern blot evidence of c-erbB-2 amplification. No evidence of activating point mutations in the c-erbB-2 protooncogene was found in any of these cases.
Base Sequence↗
Neu receptor dimerization.
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Models, Molecular↗
Expression of c-erbB-2 oncoprotein: a prognostic indicator in human breast cancer.
Sections of formalin-fixed, paraffin-embedded tissue from 185 primary breast carcinomas were stained immunohistochemically using a polyclonal antibody against the c-erbB-2 oncoprotein. Positive staining, which is known to correlate with gene amplification, was associated with earlier relapse, shorter postrelapse survival, and shorter overall survival. Lymph node, epidermal growth factor receptor, and estrogen receptor status, tumor size, and histological grade also had prognostic significance but, applying multivariate analysis, only lymph node status was a more important predictor of relapse-free and overall survival than staining for the oncoprotein. Positive staining was correlated with negative estrogen receptor status and high histological grade, but there was no association with either lymph node or epidermal growth factor receptor status or tumor size. Expression of the c-erbB-2 oncoprotein appears to be an important independent indicator of prognosis in human breast cancer.
Analysis of Variance↗
Differences in phorbol-ester-induced down-regulation of protein kinase C between cell lines.
Down-regulation of protein kinase C induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined in Swiss 3T3, V79, MDBK and C6 cells by Western blotting. Variations in the rate of down-regulation caused by treatment with 100 nM-TPA were observed; TPA treatment for 5 h caused maximal down-regulation in V79 cells, whereas TPA treatment for 10 h or 30 h was needed for maximal down-regulation of protein kinase C in MDBK or Swiss 3T3 cells respectively. The decrease in amount of immunologically detectable protein kinase C was 30% in MDBK cells and 100% in V79 and Swiss 3T3 cells. MDBK and C6 cells could be completely depleted of protein kinase C by treatment with 250 nM-TPA. In C6 cells, after treatment with 500 nM-TPA, an 80% loss of protein kinase C was seen over 10 h. Measurement of the numbers of phorbol-ester-binding sites remaining in each cell line when protein kinase C was maximally down-regulated indicated that in MDBK and Swiss 3T3 cells loss of phorbol-ester-binding sites paralleled loss of protein kinase C, whereas in V79 and C6 cells no such correlation was observed.
Animals↗
c-erbB-2 oncogene product staining in gastric adenocarcinoma. An immunohistochemical study.
The c-erbB-2 oncogene has been shown to be amplified in a variety of human adenocarcinomas. Antibodies to the protein product, p185, have been used for immunostaining of paraffin-embedded material, and have demonstrated that high levels of c-erbB-2 protein expression correlate with gene amplification under certain conditions. In studies by others, amplification has been demonstrated in 40 per cent of tubular type adenocarcinomas of the stomach, and an immunohistochemical study on frozen tissue has demonstrated staining in 3 out of 10 cases. Our study, using paraffin-embedded material, demonstrates staining in 19 per cent of 126 cases using a polyclonal antibody. Of the positive cases, 75 per cent were tubular or papillary type (P less than 0.025), and prominent staining was restricted to this group. Three cases showed well-defined positive areas in keeping with clonal expression of p185. No specific staining of normal or dysplastic epithelium adjacent to the carcinomas was found.
Adenocarcinoma↗
Expression of the c-erbB-2 proto-oncogene protein in human breast cancer.
The discovery of a dominantly transforming oncogene in ENU-induced rat CNS tumours has revealed in rats and man a gene highly related to the EGF receptor. The gene may be activated experimentally by mutation or overexpression such that its expression can convert immortalised but non-tumourigenic rat fibroblasts to a tumourigenic state. The mechanism by which this activation occurs is not known, nor is it known whether the molecule is itself a growth factor receptor. It is hypothesised, however, that in the light of its similarity to the EGF receptor it may be involved in growth regulation and that activation may occur by producing an aberrant growth control signal. The gene has been found to be amplified frequently and the protein it encodes overexpressed in a variety of human adenocarcinomas. Investigations are underway to attempt to reveal its role in these tumours and to assess its possible value as an indicator of tumour behaviour. Overexpression of the protein provides a target for tumour imaging and therapy.
Breast Neoplasms↗
c-erB-2 oncoprotein expression in malignant and nonmalignant breast tissue.
Using a specific polyclonal antibody (21N) to the c-erbB-2 oncoprotein, a total of 200 primary breast carcinomas of various histological types and 46 benign breast lesions, were immunohistochemically stained, to identify those tissues over-expressing this putative transmembrane receptor. Positivity for this oncoprotein was evident as an intense brown granular staining predominantly located at the cell membrane and was present in 17% (n = 34) of the primary breast carcinomas investigated. With the exception of one case of apocrine metaplasia, none of the remaining benign breast samples (n = 45) were positive for this oncoprotein. This study shows that overexpression of the c-erb B-2 oncoprotein is easily identifiable and can be detected on paraffin embedded sections using an immunohistochemical technique. The results obtained correlate well with other c-erbB2 detection systems and in addition, positivity for this oncoprotein is mostly confined to the malignant phenotype.
Breast Neoplasms↗
Pattern of expression of c-erbB-2 oncoprotein in human fetuses.
The pattern of expression of the c-erbB-2 oncoprotein was investigated in whole mount preparations of 11 human fetuses by immunocytochemistry using two polyclonal antibodies, 20N and 21N. c-erbB-2 was widely expressed within all three germ layers. Expression remained relatively constant in epithelial, mesodermal and extraembryonic tissues, but varied over time during the development of the fetal skeleton. Western blotting failed to detect c-erbB-2 in normal fetal tissues but did confirm expression in a microvillous membrane preparation of placenta. c-erbB-2 expression is widespread in the human fetus and occurs at an earlier stage than epidermal growth factor receptor.
Biomarkers, Tumor↗
An evaluation of immunoreactivity for c-erbB-2 protein as a marker of poor short-term prognosis in breast cancer.
Eighty-five breast carcinomas from the same number of patients have been assessed immunohistochemically using the antiserum 21N for the presence of the c-erbB-2 protein. Twenty-two of the patients had evidence of advanced disease (tumour fixation or distant metastases) at presentation. Follow-up was for a median of 24 months. c-erbB-2 protein was detected in the majority of cells in 14 (16.5%) carcinomas, and to a lesser extent in a further six (7%) tumours. There was no relationship between staining and stage, node status or size but more poorly differentiated carcinomas had evidence of staining (36%) than well (17%) or moderately (14%) differentiated carcinomas (P = 0.02). There was a significant association between staining and mortality (P = 0.009) and recurrence (P = 0.0002). The relative risk of death for staining compared to no staining (after adjusting for node status, stage and grade) was 2.97 (95% confidence interval 1.29, 6.84) and the relative risk of recurrence for staining compared to no staining after similar adjustment was 3.85 (95% confidence interval 1.86-7.97). In this particular group of patients immunoreactivity for c-erbB-2 protein is an independent indicator of poor short-term prognosis.
Biomarkers, Tumor↗
An immunohistochemical study of the presence of c-erbB-2 protein in Paget's disease of the nipple.
Tumours from 45 patients with Paget's disease of the nipple have been stained with antibody 21N which was raised against a peptide from the C-terminus of the predicted c-erbB-2 protein. Positive staining was observed in 41 cases (91%). In 42 cases the underlying mammary carcinoma was also available for study. In all but two cases staining was similar in the Paget's cells and the underlying carcinoma whether the latter was purely in situ ductal (35%) or in situ associated with infiltrating carcinoma (65%); the malignant cells in both components were large and pleomorphic and the in situ ductal carcinoma was of the comedo type. In the two exceptions the underlying carcinoma showed different histological features. The significance of the high levels of c-erbB-2 protein in this pattern of mammary carcinoma is considered.
Adult↗
Immunohistochemical detection of the c-erbB-2 proto-oncogene product in normal, benign and malignant cartilage tissues.
Benign and malignant cartilage tumours as well as normal cartilage were stained immunohistochemically with antibodies raised to a synthetic peptide from the sequence of the c-erbB-2 protein. Positive staining was present in 18/23 of the chondrosarcomas and in one case of osteochondroma. Normal and benign neoplastic cartilage tissues revealed negative results. It is concluded that expression of the c-erbB-2 protein may contribute to cell transformation in chondrosarcomas; it is not apparent whether the expression of c-erbB-2 protein in chondrosarcomas characterizes a subpopulation of different prognostic significance.
Cartilage↗
Bradykinin receptor number and sensitivity to ligand stimulation of mitogenesis is increased by expression of a mutant ras oncogene.
Bradykinin is a nine amino acid peptide of the kinin family believed to play a role in pain mediation and in the regulation of blood pressure, fluid balance and smooth muscle contraction. Here we demonstrate that bradykinin is also a potent mitogen for a mutant Ha-ras-transfected cell line, Rat 13. The Rat 13 cells display two binding sites for bradykinin: a moderate number (52,000) of high affinity sites (Kd = 4.9 nM) co-exist with a much smaller number (1100) of very high-affinity sites (Kd = 2.7 pM). Ligand binding stimulates mitogenesis through the lower affinity receptors, which are classified as B2. These receptors are down-regulated in response to ligand. In contrast, Rat 1 cells (the cell line from which Rat 13 was derived) have only 4000 receptors per cell in total and respond weakly to bradykinin as a mitogen. Thus, expression of a mutant ras protein in Rat 13 cells increases their expression of the bradykinin receptor and their sensitivity to ligand stimulation of mitogenesis. Additional binding studies demonstrate that human A431 epithelial cells and Swiss mouse 3T3 fibroblasts also possess high-affinity sites for bradykinin.
Animals↗
Immunohistochemical distribution of c-erbB-2 in infiltrating and in situ breast cancer.
An immunohistochemical study of c-erbB-2 expression was performed on invasive and in situ breast cancer. Strong membrane staining was seen in 16% of the infiltrating ductal carcinomas and 44% of the in situ lesions. c-erbB-2 was overexpressed in ductal rather than lobular tumours. Our results indicate that a small sub-group of breast carcinomas are associated with over-expression of this oncogene which may define an important subgroup of in situ and infiltrating ductal carcinomas.
Breast Neoplasms↗