Edith Blackburn

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.

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. Copyright

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 1α, Neuregulin 2α, Neuregulin 2β, Neuregulin 4 and TGFα, moderate expression was seen with EGF, Neuregulin 1β 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.

Inoculation of granular activated carbon in a fixed bed with S-triazine-degrading bacteria as a water treatment process

Abstract Two bacterial strains (SL1: Rhodococcus rhodochrous , WT1: Acinetobacter junii ) capable of biodegrading atrazine and simazine in surface water (1–10 μg l −1 ) were inoculated into fixed beds of granular activated carbon (GAC, 20 min empty bed contact time, EBCT). Eluted SL1, WT1 and indigenous bacteria were enumerated by spread plate on nutrient agar. Bacteria attached to GAC were desorbed in 50 mM Tris buffer (pH 7.5, recovery 75 ± 5%) before enumeration. Atrazine and simazine in treated water and adsorbed to GAC were extracted and determined by GC (detection limit 0.003 μg l −1 ). 48 h after inoculation with 9.8 × 10 8 SL1 or 1.3 × 10 9 WT1, 10% of the SL1 (1 ± 0.04 × 10 7 g −1 dry wt GAC) and 0.77% of the WT1 (1 ± 0.04 × 10 6 g −1 dry wt of GAC) remained in the fixed beds. No WT1 were detected in the treated water over a 28 day operating period although they were present on the GAC (2.8 ± 0.22 × 10 4 g −1 dry wt). An influent concentration of 2 mg l −1 atrazine and 2 mg l −1 simazine (in dH 2 O) was used to induce breakthrough exceeding 0.003 μg l −1 of s-triazines in treated water, and the effluent concentration of inoculated and non-inoculated columns was compared over 40 d (27 min EBCT). Up to day 18, WT1 reduced s-triazine concentration (maximum 0.15 ± 0.061 μg l −1 atrazine, 1.1 ± 0.08 μg l −1 simazine) in treated water compared to a non-inoculated column (maximum 0.49 ± 0.061 μg l −1 atrazine, 2.1 ± 0.08 μg l −1 simazine). After day 18 indigenous bacteria had acclimated to biodegrade the s-triazines and effluent concentrations were the same for both treatments. Total biodegradation of adsorbed s-triazine (87 mg atrazine and 87 mg simazine per fixed bed) ranged from 19.5 to 32% of each herbicide for both inoculated and non-inoculated GAC. Influent containing 10 μg l −1 atrazine and 10 μg l −1 simazine did not cause s-triazine breakthrough in treated water ( −1 ) over 20 days (40 min EBCT). Biodegradation of adsorbed atrazine and simazine (13.9 μg g −1 GAC for each s-triazine) by isolate SL1 (2 × 10 7 cells g −1 dry wt) was 53 ± 12.2% atrazine and 58 ± 8.2% simazine and was significantly greater than that of non-sterile, non-inoculated GAC (6 × 10 6 indigenous bacteria g −1 dry wt; 0% atrazine, 32 ± 8.2% simazine). Inoculation of GAC packed beds with s-triazine degraders reduced the amount of s-triazine adsorbed and treated water concentration within 40 days of inoculation and may extend bed life of GAC for surface water treatment.

Biodegradation of s-triazine herbicides at low concentrations in surface waters

Abstract Fourteen bacterial strains were isolated from industrial waste (SL) agricultural soil (S), surface water (B) or water treatment filter material (WT) following enrichment in a minimal salts medium (MSM) containing 5–10 mg 1−1 of both atrazine and simazine. Biodegradation by the isolates of 1 μg 1−1 of atrazine and 1 μg 1−1 simazine in 100 ml MSM or reservoir surface water was assessed by comparison with non-inoculated controls. Extraction with dichloromethane recovered 100 ± 5% of both s-triazines in non-inoculated MSM and reservoir surface water. Recovery was not reduced by adsorption of the s-triazines to bacteria, and no abiotic degradation occurred over the incubation periods. Isolate S4 degraded 20 ± 7% of the atrazine and isolates S7 and S8 degraded 40 ± 12% of the simazine in MSM after 7d at 30°C. Decreasing the NH4NO3-N in the MSM from 35 mg 1−1 to 1 mg 1−1 stimulated biodegradation of atrazine by a fourth isolate (SL3). At 10°C isolates BI, SL2, S4 and S6 each biodegraded 20 ± 1% of the atrazine and isolate SL2 biodegraded 30 ± 7% of the simazine in surface water after 15 d. No biodegradation occurred in MSM at 10°C. Granular activated carbon (GAC, 1 g 1−1) was added to surface water (10 μg 1−1 atrazine and 10 μg 1−1 simazine) which adsorbed 9.7 μgg−1 s-triazine and provided sites for bacterial attachment. Recovery of adsorbed s-triazines (1, 10 and 40 μg 1−1) by Soxhlet extraction of non-inoculated GAC was 97 ± 1.5%. Inoculation of s-triazine-degraders into surface water containing GAC reduced the solution concentration (0.3 μg 1−1 atrazine and 0.3 μg 1−1 simazine) by up to 86 ± 18% (S8) after incubation for 15 d at 10°C. Biodegradation of 25 μg 1−1 atrazine and 28 μg 1−1 simazine in surface water amended with 0.5 g 1−1 GAC and inoculated with isolate WT1 was 50 ± 4% (atrazine) and 38 ± 4% (simazine) after 21 d at 10°C. Biodegradation by WT1 attached to 0.5 g GAC in surface water (0.5 μg 1−1 atrazine and 6 μg 1−1 simazine) was increased from zero atrazine and 23 ± 7.6% simazine to 40 ± 5.3% atrazine and 71 ± 5.6% simazine following transfer of bioaugmented GAC into fresh surface water. Inoculation of GAC filters with selected strains has potential as a biotreatment for surface water containing s-triazine herbicides.

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.

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.

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.

Abstract 3445: The role of the truncated product of the neuregulin 4 gene in cancer and normal tissues

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The Epidermal Growth Factor Receptor (EGFR) family consists of a group of four transmembrane proteins, EGFR, the Human Epidermal Growth Factor 2 (HER2), HER3 and HER4 that are each involved in cell-signalling. They are activated by ligands, which include the neuregulin (NRG) family of proteins. These proteins are produced as various splice variants from four genes, NRG1-4. Although NRG1 has been extensively investigated, not much is known about the other neuregulins. Thus far, the NRG4 gene has been found to encode five proteins, NRG4 A1, NRG4 A2, NRG4 B1, NRG4 B2 and NRG4 B3. NRG4s B1-3 are truncated structures, which either prematurely terminate as in NRG4s B1 and B2, or have an additional 21 amino acids as in NRG4 B3. Little is known about the structure and function of the NRG4 B3. Previous studies have suggested that it has a different disulphide bond arrangement than that of the other EGF-like ligands. In this study, we have surveyed the expression of NRG4 B3 in breast, lung and colorectal cancers, and found that it is expressed at variable levels. Nuclear magnetic resonance spectroscopy of re-folded NRG4 B3 suggests it has a largely unfolded structure very different from the typical EGF-like molecules. It also has distinct properties in that it is a weak agonist for HER4 signaling and inhibits rather than promotes cell motility in various model systems. Taken together, this suggests that it may have a distinct role in normal and malignant tissues differing in properties to typical EGF-like proteins. Citation Format: Markella J. Alatsatianos, Edith Blackburn, William Gullick. The role of the truncated product of the neuregulin 4 gene in cancer and normal tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3445. doi:10.1158/1538-7445.AM2014-3445