[Show abstract][Hide abstract] ABSTRACT: Brown adipocytes dissipate energy whereas white adipocytes are an energy storage site. We explored the plasticity of different white adipose tissue depots in acquiring a brown phenotype by cold exposure. By comparing cold-induced genes in white fat to those enriched in brown compared to white fat, at thermoneutrality, we defined a "BRITE" transcription signature. We identified the genes, pathways and promoter regulatory motifs associated with "browning" as these represent novel targets for understanding this process. For example, Neuregulin 4 was more highly expressed in brown adipose tissue, upregulated in white fat upon cold exposure and cell studies showed it is a neurite outgrowth-promoting adipokine, indicative of a role in increasing adipose tissue innervation in response to cold. A cell culture system that allows us to reproduce the differential properties of the discrete adipose depots was developed to study depot specific differences at an in vitro level. The key transcriptional events underpinning white adipose tissue to brown transition are important as they represent an attractive proposition to overcome the detrimental effects associated with metabolic disorders including obesity and Type 2 diabetes.
AJP Endocrinology and Metabolism 02/2014; · 4.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Islet cell growth and function are affected by ligands from the Epidermal Growth Factor family. We describe here the expression, regional distribution and effect on growth and secretion of insulin of a subset of these, the Neuregulin family. The expression of NRG1 alpha, NRG1 beta, NRG2 alpha, NRG2 beta, NRG3 and NRG4 in rat islets was determined using immunohistochemical and double immunofluorescent staining. We also report the expression of the four receptors and the remaining seven ligands using immunohistochemistry. The NRG1 alpha splice variant was expressed in beta cells and the NRG1 beta variant mainly in alpha cells. NRG3 was also predominantly present in alpha cells. Most of the members of the EGF family of ligands were also expressed, with Epigen being present at the highest levels. The rat islet derived cell line CRI-G1 was used to study the effect of addition of EGF, NRG1 beta, NRG3 and NRG4 on cell growth and insulin secretion. Synthetic refolded NRG3 strongly stimulated the growth of the CRI-G1 cells and NRG4 gave the greatest stimulation of insulin release. Different members of the Neuregulin family are therefore potentially potent stimuli for islet cell growth and insulin release and differ in expression in alpha and beta cells.
[Show abstract][Hide abstract] 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. · 4.47 Impact Factor
[Show abstract][Hide abstract] 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. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The neuregulin 4 gene encodes at least five different variants (designated A1, A2, B1, B2 and B3) produced as a result of alternative splicing. We have determined their sites of expression in normal human adult tissues using isoform-specific antibodies. Their expression is cell type specific and differs in subcellular location suggesting that they may have varied functions in these contexts. We have shown in a panel of prostate cancers that each form is present to differing degrees, and that principal component analysis indicates that there are three patterns of expression. Some isoforms were positively correlated with high prostate-specific antigen levels and others were inversely associated with Gleason score. Synthetic, refolded A forms promoted lamellipodia and filopodia formation in cells expressing the ErbB4 (CTa) receptor and stimulated cell motility in wound healing assays. The data suggest that the different forms have varied sites of expression and function, and this includes effects on cell architecture and motility.
Endocrine Related Cancer 10/2010; 18(1):39-49. · 5.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The levels of expression of the four receptors and eleven ligands composing the epidermal growth factor family were measured using immunohistochemical staining in one hundred cases of breast cancer. All of the family were expressed to some degree in some cases; however, individual cases showed a very wide range of expression of the family from essentially none to all the factors at high levels. The highest aggregate level of expression of a receptor was HER2 followed by HER1, then HER3, then HER4. The ligands (including two splice variants of the NRG1 and NRG2 genes) broadly fell into three groups, those with the highest aggregate expression were Epigen, Epiregulin, Neuregulin 1alpha, Neuregulin 2alpha, Neuregulin 2beta, Neuregulin 4 and TGFalpha, moderate expression was seen with EGF, Neuregulin 1beta and Neuregulin 3, and relatively low levels of expression were seen of HB-EGF, Betacellulin and Amphiregulin. Statistical analysis using Spearman's Rank Correlation showed a positive correlation of expression between each of the factors. Analysing the data using the Cox Proportional Hazards model showed that, in this dataset, the most powerful predictors of relapse free interval and overall survival were the combined measurement of only Epigen and Neuregulin 4.
Breast Cancer Research and Treatment 09/2009; 122(1):105-10. · 4.47 Impact Factor
[Show abstract][Hide abstract] 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. · 5.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: This chapter gives an overview of computational models and simulations of the EGF receptor system. It begins with a survey
of motivations for producing such models and then describes the main approaches that are taken to carrying out such modeling,
with respect to differential equations and individual-based modeling. Finally, a number of projects that apply modeling and
simulation techniques to various aspects of the EGF receptor system are described.
KeywordsEGF receptor–computational models–mathematical models
[Show abstract][Hide abstract] 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. · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
British Journal of Cancer 02/2007; 96(2):284-9. · 4.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two synthetic peptides from the predicted sequence of the human c-erbB-2 protein were synthesized and used to raise antisera in rabbits. The sequences chosen were identical to those in the homologous rat c-neu protein. The antibodies produced reacted with the immunizing peptides in ELISA but showed little or no cross-reaction with the partly homologous peptides found in equivalent positions in the human EGF receptor. Both antipeptide antibodies, and a monoclonal antibody (MAb) specific for the rat new protein, immuno-precipitated a 185-kDa protein from 35S-methionine-labelled lysates prepared from a rat cell line known to express high levels of the c-neu protein. The antipeptide antibodies also recognized a protein of the same size in Western blots. In addition, both antipeptide antibodies immunoprecipitated a 190-kDa protein from labelled cell lysates prepared from human and monkey cells. Antibodies to one of the peptides, which showed no detectable cross-reaction with human, rat or monkey EGF receptor, were used to examine the expression of the c-erbB-2 protein on a variety of cultured cell types. Eleven transformed, I non-established and 2 immortalized cell types were examined by immunoprecipitation for their level of expression of the c-erbB-2 protein and of the EGF receptor. The numbers of EGF receptors varied widely between different cell lines, whereas the level of the c-erbB-2 protein, which was found on all of the cell types examined, was more constant. The number of c-erbB-2 molecules present was estimated by autoradiography to be about 100,000 per cell. The antibodies were then used to examine the location and level of expression of the human c-erbB-2 and rat c-neu proteins in normal tissues. Immunohistochemical staining showed that the c-erbB-2 protein was highly expressed in rat kidney proximal tubules and loop of Henle. The c-enbB-2 protein was also present on normal human epithelial cells but in some cases with a different distribution to that of the EGF receptor.
International Journal of Cancer 07/2006; 40(2):246 - 254. · 6.20 Impact Factor
[Show abstract][Hide abstract] 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. · 4.91 Impact Factor
[Show abstract][Hide abstract] 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. · 4.20 Impact Factor