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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

Mapping nucleolar and spliceosome localization sequences of neuregulin1-beta3.

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.

Co-expression of neuregulins 1, 2, 3 and 4 in human breast cancer.

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.

c-erbB-4/HER4: friend or foe?

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.

A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) has attracted considerable attention as a target for cancer therapy. Wild-type (wt)EGFR is amplified/overexpressed in a number of tumor types, and several mutant forms of the coding gene have been found, with DeltaEGFR, a deletion mutation lacking exons 2-7 of the external domain, being the most common and particularly associated with glioblastoma. We generated monoclonal antibodies (mAbs) against NR6(DeltaEGFR) (mouse fibroblast line NR6 transfected with DeltaEGFR). mAb 806 with selective reactivity for NR6(DeltaEGFR) in mixed hemadsorption assays, fluorescence-activated cell sorting, Western blot, and immunohistochemistry was analyzed in detail and compared with mAbs 528 (anti-wtEGFR) and DH8.3 (anti-DeltaEGFR). In xenograft tumors and molecularly pretyped glioblastomas, the reactivity pattern was as follows: 528 reactive with amplified and nonamplified wtEGFR; DH8.3 reactive with DeltaEGFR; and 806 reactive with amplified/overexpressed wtEGFR (with or without DeltaEGFR). In normal tissues, 528 but not DH8.3 or 806 was widely reactive with many organs, e.g., liver expressing high EGFR levels. In glioblastoma and non-CNS tumor panels, 806 was reactive with a high proportion of glioblastomas and a substantial number of epithelial cancers of lung and of head and neck. DH8.3 reactivity was restricted to DeltaEGFR-positive glioblastoma. Thus, 806 represents a category of mAbs that recognizes tumors with EGFR amplification/overexpression but not normal tissues or tumors with normal EGFR levels. Our study also indicates that DeltaEGFR is restricted to glioblastoma, in contrast to other reports that this mutation is found in tumors outside the brain.