Brown and white adipose tissues: intrinsic differences in gene expression and response to cold exposure in mice.

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 with 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 and upregulated in white fat upon cold exposure, and cell studies showed that 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.

A monoclonal antibody to the human HER3 receptor inhibits Neuregulin 1-beta binding and co-operates with Herceptin in inhibiting the growth of breast cancer derived cell lines.

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.

Localisation of Neuregulin 1-ß3 to different sub-nuclear structures alters gene expression.

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-ß3 (NRG1-ß3) isoform localises to two sub-nuclear compartments in animal cells, nucleoli and spliceosomes. We used NRG1-ß3 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-ß3 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-ß3 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.

The complete family of epidermal growth factor receptors and their ligands are co-ordinately expressed in breast cancer.

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.

Identification and characterization of novel spliced variants of neuregulin 4 in prostate cancer.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSION: 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.

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.

Epidermal growth factor receptor tyrosine kinase inhibitors and bone metastases: different mechanisms of action for a novel therapeutic application?

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.

A recurrent chromosome breakpoint in breast cancer at the NRG1/neuregulin 1/heregulin gene.

Most studies of genomic rearrangements in common cancers have focused on regional gains and losses, but some rearrangements may break within specific genes. We previously reported that five breast cancer cell lines have chromosome translocations that break in the NRG1 gene and that could cause abnormal NRG1 expression. NRG1 encodes the Neuregulins 1 (formerly the Heregulins), ligands for members of the ErbB/epidermal growth factor-receptor family, which includes ErbB2/HER2. We have now screened for breaks at NRG1 in paraffin sections of breast tumors. Tissue microarrays were screened by fluorescence in situ hybridization, with hybridization probes proximal and distal to the expected breakpoints. This screen detects breaks but does not distinguish between translocation or deletion breakpoints. The screen was validated with array-comparative genomic hybridization on a custom 8p12 high-density genomic array to detect a lower copy number of the sequences that were lost distal to the breaks. We also precisely mapped the breaks in five tumors with different hybridization probes. Breaks in NRG1 were detected in 6% (19 of 323) of breast cancers and in some lung and ovarian cancers. In an unselected series of 213 cases with follow-up, breast cancers where the break was detected tended to be high-grade (65% grade III compared with 28% of negative cases). They were, like breast tumors in general, mainly ErbB2 low (11 of 13 were low) and estrogen receptor positive (11 of 13 positive).

Heparin-binding epidermal growth factor and its receptors mediate decidualization and potentiate survival of human endometrial stromal cells.

Heparin-binding epidermal growth factor (HB-EGF) has pleiotropic biological functions in many tissues, including those of the female reproductive tract. It facilitates embryo development and mediates implantation and is thought to have a function in endometrial receptivity and maturation. The mature HB-EGF molecule manifests its activity as either a soluble factor (sol-HB-EGF) or a transmembrane precursor (tm-HB-EGF) and can bind two receptors, EGFR and ErbB4/HER4. In this study, we identify factors that modulate expression of HB-EGF, EGFR, and ErbB4 in endometrial stromal cells in vitro. We demonstrate that levels of sol- and tm-HB-EGF, EGFR, and ErbB4 are increased by cAMP, a potent inducer of decidualization of the endometrial stroma. We also show that production of sol- and tm-HB-EGF is differentially modulated by TNF alpha and TGF beta. Our data suggest that HB-EGF has a function in endometrial maturation in mediating decidualization and attenuating TNF alpha- and TGF beta-induced apoptosis of endometrial stromal cells.

Simulating complex intracellular processes using object-oriented computational modelling.

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.

Heparin-binding epidermal growth factor and its receptor ErbB4 mediate implantation of the human blastocyst.

The mechanisms that mediate implantation of the human embryo remain poorly understood and represent a fundamental problem in reproductive biology. Candidate molecules that mediate and facilitate implantation have been identified in animal studies, and include heparin binding epidermal growth factor. Here we demonstrate a potential function for the transmembrane form of heparin-binding epidermal growth factor in mediating blastocyst attachment to the endometrium, in two different novel in vitro models for human implantation. Furthermore, we demonstrate specific localisation of the heparin-binding epidermal growth factor receptor ErbB4, on the surface of the trophectoderm in peri-implantation human blastocysts. Our data lead the way for further dissection of the molecular mechanisms of implantation of the human embryo, and have implications for infertility, in vitro fertilization and contraception.

Neuregulin expression, function, and signaling in human ovarian cancer cells.

PURPOSE: To investigate the expression and function of neuregulin (NRG) isoforms in ovarian cancer cell lines and tumor samples. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

The mitogenic potential of heparin-binding epidermal growth factor in the human endometrium is mediated by the epidermal growth factor receptor and is modulated by tumor necrosis factor-alpha.

Heparin-binding epidermal growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is implicated in a variety of biological processes, including reproduction. Previous studies describe increased levels of HB-EGF in the human endometrium during the midsecretory stage of the menstrual cycle, suggesting a function for HB-EGF in implantation of the human blastocyst. Here we have investigated the expression and function of the soluble and transmembrane forms of HB-EGF in the human endometrium. We show that the expression of the transmembrane form of HB-EGF in the human endometrium is modulated according to the stage of the menstrual cycle. We present data demonstrating that both the soluble and transmembrane forms of HB-EGF induce DNA synthesis in human endometrial stromal cells. Furthermore, TNFalpha has a cooperative effect on HB-EGF, EGF, TGFalpha, and betacellulin-induced DNA synthesis in stromal cells, suggesting roles for the EGF family and TNFalpha in regeneration and maturation of human endometrium. Induction of DNA synthesis by HB-EGF and its modulation by TNFalpha in endometrial stromal cells are mediated by the EGF receptor and not the HB-EGF receptor ErbB4. Our data suggest key functions for HB-EGF, TNFalpha, and the EGF receptor in endometrial maturation, via autocrine/paracrine and juxtacrine pathways, in preparation for embryo implantation.

Transforming growth factor alpha, amphiregulin and cripto-1 are frequently expressed in advanced human ovarian carcinomas.

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.

The ErbB4 CYT2 variant protects EGFR from ligand-induced degradation to enhance cancer cell motility.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor-stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER+ HER2- breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.

The neuregulin system of ligands and their receptors in rat islets of langerhans.

Islet cell growth and function are affected by ligands from the epidermal growth factor (EGF) family. We describe here the expression, regional distribution, and effect on growth and secretion of insulin of a subset of these, the neuregulin (NRG) family. The expression of NRG1α, NRG1ß, NRG2α, NRG2ß, NRG3, and NRG4 in rat islets was determined using immunohistochemical and double immunofluorescent staining. We also report the expression of the 4 receptors and the remaining 7 ligands using immunohistochemistry. The NRG1α splice variant was expressed in ß-cells and the NRG1ß variant mainly in α-cells. NRG3 was also predominantly present in α-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, NRG1ß, 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 NRG family are therefore potentially potent stimuli for islet cell growth and insulin release and differ in expression in α- and ß-cells.

Expression of neuregulin 4 splice variants in normal human tissues and prostate cancer and their effects on cell motility.

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.

The neuregulin family of genes and their multiple splice variants in breast cancer.

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.

The role of the epidermal growth factor receptor in breast cancer.

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.

Neuregulins 1-4 are expressed in the cytoplasm or nuclei of ductal carcinoma (in situ) of the human breast.

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.