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ABSTRACT: The HER3 protein contributes to malignant transformation in breast and other cancer types as a consequence of elevated levels of expression, particularly in the presence of the HER2 protein. We show here that an antibody, called SGP1, to the extracellular domain of the HER3 receptor can inhibit completely Neuregulin stimulated growth of cultured breast cancer cells. Herceptin is a humanised monoclonal antibody to the HER2 protein which has an established role in the treatment of some patients with breast cancer. We demonstrate that Herceptin and SGP1 can bind simultaneously to breast cancer cells expressing both the HER2 and HER3 proteins. In the presence of moderate levels of Herceptin, addition of the SGP1 monoclonal antibody gave a dose-dependent inhibition of the growth of cells expressing both the high levels and moderate levels of HER2. The combination of Herceptin with SGP1 is effective in inhibiting breast cancer cell growth in cases where both HER2 and HER3 are expressed.
Breast Cancer Research and Treatment 12/2011; 134(1):53-9. · 4.43 Impact Factor
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ABSTRACT: Neuregulins are growth factors that signal via the ErbB3 and ErbB4 receptors. Here we show using immunohistochemistry that they are often expressed in the nucleus of a range of tumour types including soft tissue and breast. The Neuregulin 1 type I-β3 (NRG1-β3) isoform localises to two sub-nuclear compartments in animal cells, nucleoli and spliceosomes. We used NRG1-β3 tagged with photoactivatable GFP and demonstrated that this re-localised from nucleoli to spliceosomes over 90 min. Tyrosine kinase activity was not required for retaining the NRG1-β3 within the nucleus. Mutation of the lysines 14 and 16 or 15 and 16 together prevented nucleolar uptake while four positively charged residues were identified which were required for spliceosome uptake. Molecular modelling suggests that three of these may form a binding site. We showed using a kinome array that NRG1-β3 and a mutant exclusively localising to spliceosomes increased phosphorylation and/or expression of the HER4 and HER2 receptors. Using a transcriptomic analysis the same two constructs induced expression of several messenger RNAs and we confirmed the increased expression at the protein level of the most highly induced, Heat Shock Protein 70B'. These results suggest that Neuregulin activates receptor signalling in spliceosomes leading to altered gene expression.
Experimental Cell Research 02/2011; 317(4):423-32. · 3.58 Impact Factor
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William J Gullick
European journal of cancer (Oxford, England: 1990) 09/2009; 45 Suppl 1:205-10. · 4.12 Impact Factor
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ABSTRACT: The neuregulin family consists of four genes, NRG1-4 which can each encode products containing a domain related to the epidermal growth factor family of ligands. Each gene is subject to complex control of transcription and to splicing of their mRNA product to give many variant proteins. These do not contain secretory sequences but some, through their transmembrane sequence, are routed via the Golgi where they are glycosylated, to the cell surface. Here they may be released by regulated proteolysis to act as soluble proteins which can interact and activate members of the EGF receptor family of receptor tyrosine kinases. Other splice variants do not encode transmembrane sequences and these are found either in the cytoplasm or, if they encode a nuclear localisation sequence, in distinct compartments in the nucleoplasm. It has been shown that the variants containing a full EGF domain can act as receptor agonists but the function of the cytoplasmic and nuclear products is unknown as yet. All four neuregulin genes are expressed and play an important role in mammary gland development. They are also expressed at elevated levels in some cases of ductal carcinoma in situ of the breast and breast cancer. They seem to be active in this setting and their presence may affect the efficacy of treatment with endocrine agents or with signal transduction inhibitors directed at the EGF receptor family members. Much remains to be learned however of their normal function and their influence on breast cancer development, progression and response to therapy.
Journal of Mammary Gland Biology and Neoplasia 07/2008; 13(2):205-14. · 6.74 Impact Factor
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Journal of Mammary Gland Biology and Neoplasia 07/2008; 13(2):157. · 6.74 Impact Factor
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ABSTRACT: The neuregulin (NRG) 1, 2, and 3 genes undergo extensive alternative mRNA splicing, which results in variants that show structural and functional diversity. The aims of this study were to establish whether the fourth member of this family, NRG4, is expressed in prostate cancer, if it is alternatively spliced and whether any functional differences between the variants could be observed.
The expression of NRG4 was determined using immunohistochemical staining of 40 cases of primary prostate cancer. Bioinformatic analysis and reverse transcription-PCR (RT-PCR) using NRG4 isotype-specific primers on a panel of normal and prostate cancer cell lines were used to identify alternatively spliced NRG4 variants. Expression of these variants was determined using isotype-specific antibodies. Transfection into Cos-7 cells of two of these green fluorescent protein-tagged variants allowed analysis of their subcellular location. Four of the variants were chemically synthesized and tested for their ability to activate the ErbB4 receptor.
NRG4 was variably expressed in the cytoplasm in the majority of prostate cancer cases, and in a subset of cases in the membrane, high levels were associated with advanced disease stage. Four novel NRG4 splice variants (NRGA2, NRG4 B1-3) were characterized, where each seemed to have a different subcellular location and were also expressed in the cytoplasm of the prostate tumors. NRG4 B3 was also present in endothelial cells. In transfected cells, the A type variant (NRG4 A1) was localized to the membrane, whereas the B type variant (NRG4 B1), which lacks the predicted transmembrane region, had an intracellular localization. Only the variants with an intact epidermal growth factor-like domain activated ErbB4 signaling.
NRG4 overexpression is associated with advanced-stage prostate cancer. The alternative splice variants may have different roles in cell signaling, some acting as classic receptor ligands and some with as-yet unknown functions.
Clinical Cancer Research 07/2007; 13(11):3147-55. · 7.74 Impact Factor
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ABSTRACT: A new family of epidermal growth factor-like proteins, the Neuregulins (NRGs), have recently been identified and are expressed in a range of normal tissues and in some forms of cancer including breast cancer. In this study we examined using immunohistochemical staining expression of NRG1alpha, NRG1beta, NRG2alpha, NRG2beta, NRG3 and NRG4 in sixty cases of pre-invasive ductal carcinoma in situ of the breast representing different degrees of differentiation. Each protein was expressed in a high proportion of these cases showing a predominantly homogenous cytoplasmic staining pattern. Nuclear expression of NRG1alpha, NRG1beta, and NRG3 was however also observed in a significant fraction of cases. High levels of expression of NRG2beta and NRG4 were associated with high-grade tumours (p< or =0.005), NRG2beta staining was associated with tumour size >25 mm (p=0.005) while NRG3 nuclear staining was present more often in low-grade tumours (p=0.039). This data demonstrates that each member of the NRG family of ligands is present in pre-invasive ductal breast cancer and that they may be involved in regulating cell behaviour. The significance of intranuclear expression remains to be determined but suggests a novel mechanism of action for some of these proteins.
Breast Cancer Research and Treatment 04/2006; 96(2):163-8. · 4.43 Impact Factor
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ABSTRACT: The paper by Angelucci et al. published in the current issue of Endocrine-Related Cancer suggests a potential, novel application of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in the treatment of bone metastases. Interestingly, activity of anti-EGFR agents on the pathogenesis and progression of bone metastases has been described in previous reports, and a number of different mechanisms seem to be involved in this phenomenon. Anti-EGFR agents have a direct activity on tumour cells in which they produce growth inhibition, apoptosis, and reduced invasive capacity through the inhibition of molecules associated with tissue invasion such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9. In addition, these compounds have an anti-angiogenic activity, either direct by affecting the proliferation and survival of endothelial cells, or indirect by blocking the production of vascular endothelial growth factor (VEGF) in bone marrow stromal cells and in tumour cells. Finally, EGFR-TKIs can inhibit recruitment of osteoclasts in bone lesions, by affecting the ability of bone marrow stromal cells to induce osteoclast differentiation and activation. Taken together, these findings strongly support prospective clinical trials of anti-EGFR agents in cancer patients with bone metastases in order to define their role in the management of bone disease.
Endocrine Related Cancer 04/2006; 13(1):3-6. · 4.36 Impact Factor
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ABSTRACT: Recent trials of drug therapy targeting the erbB receptor HER2 have met with success in breast cancer. The epidermal growth factor receptor or EGFR is a closely related receptor from this same family that is involved in cellular signal transduction and tumor cell growth and survival. Emerging evidence indicates that EGFR is implicated in the development of hormone-resistant breast cancer, and that its activity is intertwined with estrogen receptor. Here, the role of EGFR in breast cancer is reviewed, and data from selected clinical trials of signal transduction inhibition of this cellular target are summarized.
Journal of Mammary Gland Biology and Neoplasia 02/2006; 11(1):3-11. · 6.74 Impact Factor
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ABSTRACT: The aim of this paper is to give an overview of computer modelling and simulation in cellular biology, in particular as applied to complex biochemical processes within the cell. This is illustrated by the use of the techniques of object-oriented modelling, where the computer is used to construct abstractions of objects in the domain being modelled, and these objects then interact within the computer to simulate the system and allow emergent properties to be observed. The paper also discusses the role of computer simulation in understanding complexity in biological systems, and the kinds of information which can be obtained about biology via simulation.
Progress in Biophysics and Molecular Biology 12/2004; 86(3):379-406. · 3.20 Impact Factor
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ABSTRACT: Mitogenic growth factors are generally cell surface associated or secreted proteins, which produce effects by binding to cell surface receptor tyrosine kinases. More recently, it has become clear that some of these proteins can accumulate in the nucleus, where they are proposed to have transcriptional activity. We show here that neuregulin1 (NRG1-beta), an EGF-like growth factor, localizes to the cell nuclei of a human breast cancer. We also show that a nonsecreted isoform of this family of ligands, neuregulin1-beta3, localizes to two distinct compartments within the nucleus, nucleoli, and SC35-positive speckles. Importantly, localization of NRG-beta3 to either structure is receptor-independent, as it occurs in cells lacking its cognate receptors, erbB-3 and erbB-4, and is unaffected by removal of the receptor-binding domain. A panel of deletion mutants was used to demonstrate that the first 21 amino acids of the N-terminus are essential for nucleolar localization, while targeting to nuclear speckles requires residues 49-79 of the 241 amino acid protein. These observations support the idea that secretion and subsequent cell surface receptor binding of mitogenic growth factors are not a prerequisite for nuclear localization and that nonsecreted ligands may have highly specific functions in defined nuclear compartments.
Experimental Cell Research 10/2004; 299(1):110-8. · 3.58 Impact Factor
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ABSTRACT: We have produced antibodies to the NRG2-alpha, NRG2-beta, NRG3 and NRG4 proteins and used these, and previously described antibodies to NRG1-alpha and NRG1-beta, to detect expression of each ligand by immunocytochemical staining in a series of 45 breast cancers. Each protein was expressed in a proportion of cases. Statistical analysis suggested that expression of one factor was associated with a high probability that other members of the family were co-expressed. NRG2-alpha expression was associated with node positivity (p-value = 0.005). The mRNAs for NRG1, 2, 3 and 4 were found in established breast cancer cell lines and NRG1, 2 and 3 mRNAs were detected in primary breast cancers. Expression of NRG4 mRNA was shown by in situ hybridization in sections from primary breast cancers. This data demonstrates that each member of the NRG family of ligands is expressed in breast cancer and suggests that they may be involved in regulating cell behaviour.
The Journal of Pathology 07/2004; 203(2):672-80. · 6.32 Impact Factor
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Scientific Programming. 01/2004; 12:25-43.
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ABSTRACT: This paper describes ongoing work on a project to simulate the behaviour of epidermal growth factor receptors. These are structures which can be found on the surface of cells in the body, which receive and process chemical signals concerned with cell growth. The implementation of a program which simulates the stimulation and clustering behaviour of these structures is described, then the paper discusses how the simulation can be scaled up so that a whole cell can be simulated on a tractable timescale. Finally some some early results are given which show the effect of changing parameters in the system, and discuss ongoing work on calibrating the simulation against results from experiments.
08/2003;
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William J Gullick
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ABSTRACT: The c-erbB-4 receptor has ligand stimulated tyrosine kinase activity but reports differ as to whether it induces cell division or differentiation. There is also controversy regarding its role in breast cancer, with some reports associating overexpression with short survival and others with longer life.
The Journal of Pathology 08/2003; 200(3):279-81. · 6.32 Impact Factor
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ABSTRACT: To investigate the expression and function of neuregulin (NRG) isoforms in ovarian cancer cell lines and tumor samples.
Expression of NRG-1alpha and NRG-1beta proteins were detected by immunohistochemistry and mRNA by RT-PCR. erbB receptor levels and downstream signaling proteins were measured by Western blot analysis.
Expression of NRG-1alpha and NRG-1beta proteins were detected by immunohistochemistry in 46 of 53 (87%) and 41 of 53 (77%) ovarian carcinomas, respectively. Serous carcinomas express higher levels of NRG-1alpha than endometrioid carcinomas (P = 0.017). NRG mRNA was detected by RT-PCR in 20 of 24 (83%) of ovarian carcinomas and eight of nine (89%) ovarian cancer cell lines. NRG-1alpha stimulated the growth of 5 of 14 cell lines whereas NRG-1beta stimulated 7 of 14 cell lines. The magnitude of NRG growth response was significantly associated with erbB2 expression levels. NRG-1alpha and -1beta (1 nM) growth-stimulated cell lines PE01 and PE06 demonstrated increased tyrosine phosphorylation of erbB2 and elevated tyrosine phosphorylation of ERK1 and ERK2. In contrast, the SKOV-3 cell line, the growth of which was unaffected, did not show these downstream responses. An anti-erbB3 receptor antibody (clone H3.105.5) blocked NRG-1beta growth changes and signaling in these cell lines. Conversely, the anti-erbB4 antibody (clone H4.72.8) enhanced NRG-beta1 growth stimulation. Herceptin also inhibited growth.
With NRG expression in the majority of ovarian carcinomas and cell lines, there is the potential for autocrine regulation of cell growth. Interfering with ligand-receptor interactions by receptor blocking antibodies suggests erbB3 is primarily involved in NRG-1beta-induced proliferation, with erbB4 having a more complex role.
Clinical Cancer Research 01/2003; 8(12):3933-42. · 7.74 Impact Factor
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Antonio D'Antonio,
Simona Losito,
Sandro Pignata,
Michele Grassi,
Francesco Perrone,
Antonella De Luca,
Rosa Tambaro,
Caterina Bianco, William J Gullick,
Gibbes R Johnson,
Vincenzo R Iaffaioli,
David S Salomon,
Nicola Normanno
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ABSTRACT: The expression of transforming growth factor alpha (TGFalpha), amphiregulin (AR) and cripto-1 (CR-1) was assessed by immunohistochemistry in 83 specimens (59 primary ovarian tumors and 24 extra-ovarian carcinomas) that were obtained from 68 ovarian carcinoma patients. Within the 59 primary tumors, 54 (92%) expressed immunoreactive TGFalpha, 45 (76%) expressed AR, and 28 (47%) expressed CR-1. The expression of AR and CR-1 mRNAs in the ovarian carcinomas was also demonstrated by RT-PCR analysis. Seventeen extra-ovarian specimens (71%) were found to express CR-1, whereas AR and TGFalpha were expressed respectively in 21 (87%) and 22 (92%) extra-ovarian tissues. In 15 cases for whom both ovarian and extra-ovarian tissues were available, a statistically significant higher expression of CR-1 was found in extra-ovarian specimens. A statistically significant correlation was found between AR expression in the ovarian carcinomas and both low grade and low proliferative activity. Finally, expression of TGFalpha was predictive of longer progression-free survival. These data strongly suggest that the EGF-related peptides might be involved in the pathogenesis and outcome of human ovarian cancer.
International Journal of Oncology 12/2002; 21(5):941-8. · 2.40 Impact Factor
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Proceedings of the 2002 ACM Symposium on Applied Computing (SAC), March 10-14, 2002, Madrid, Spain; 01/2002
