Development of a novel 7 immune-related genes prognostic model for oral cancer: A study based on TCGA database.

Oral squamous cell carcinoma (OSCC) is an aggressive tumor whose prognosis has little improvement in the last three decades. Various immune-related genes have been suggested as significant roles in the development and progression of malignant cancers. In this study, we acquired and integrated differentially expressed genes of OSCC patients, including immune-related genes and transcription factors (TFs), from The Cancer Genome Atlas (TCGA) database. TF-mediated network was established to exploring the regulatory mechanisms of prognostic immune-related genes. A 7 immune-related genes prognostic model for OSCC was obtained, including CGB8, CTLA4, TNFRSF19, CCL26, NRG1, TPM2 and PLAU, which was further proved to be an independent prognostic indicator after adjusting for other clinical factors. The immune-related genes prognostic index was significantly negatively correlated to the infiltration abundances of B cells (P < 0.05) and CD8+ T cells (P < 0.05). The novel proposed immune-based prognostic model not only provided a promising biomarker and a way to monitor the long-term treatment of OSCC, but also gave a new insight into a potential immunotherapy strategy.

Heregulin (HRG) assessment for clinical trial eligibility testing in a molecular registry (PRAEGNANT) in Germany.

BACKGROUND: Eligibility criteria are a critical part of clinical trials, as they define the patient population under investigation. Besides certain patient characteristics, clinical trials often include biomarker testing for eligibility. However, patient-identification mostly relies on the trial site itself and is often a time-consuming procedure, which could result in missing out on potentially eligible patients. Pre-selection of those patients using a registry could facilitate the process of eligibility testing and increase the number of identified patients. One aim with the PRAEGNANT registry (NCT02338167) is to identify patients for therapies based on clinical and molecular data. Here, we report eligibility testing for the SHERBOC trial using the German PRAEGNANT registry. METHODS: Heregulin (HRG) has been reported to identify patients with better responses to therapy with the anti-HER3 monoclonal antibody seribantumab (MM-121). The SHERBOC trial investigated adding seribantumab (MM-121) to standard therapy in patients with advanced HER2-negative, hormone receptor-positive (HR-positive) breast cancer and HRG overexpression. The PRAEGNANT registry was used for identification and tumor testing, helping to link potential HRG positive patients to the trial. Patients enrolled in PRAEGNANT have invasive and metastatic or locally advanced, inoperable breast cancer. Patients eligible for SHERBOC were identified by using the registry. Study aims were to describe the HRG positivity rate, screening procedures, and patient characteristics associated with inclusion and exclusion criteria. RESULTS: Among 2769 unselected advanced breast cancer patients, 650 were HER2-negative, HR-positive and currently receiving first- or second-line treatment, thus potentially eligible for SHERBOC at the end of current treatment; 125 patients also met further clinical eligibility criteria (e.g. menopausal status, ECOG). In the first/second treatment lines, patients selected for SHERBOC based on further eligibility criteria had a more favorable prognosis than those not selected. HRG status was tested in 38 patients, 14 of whom (36.8%) proved to be HRG-positive. CONCLUSION: Using a real-world breast cancer registry allowed identification of potentially eligible patients for SHERBOC focusing on patients with HER3 overexpressing, HR-positive, HER2-negative metastatic breast cancer. This approach may provide insights into differences between patients eligible or non-eligible for clinical trials. TRIAL REGISTRATION: Clinicaltrials, NCT02338167 , Registered 14 January 2015 - retrospectively registered.

Avidity-Based Affinity Enhancement Using Nanoliposome-Amplified SPR Sensing Enables Low Picomolar Detection of Biologically Active Neuregulin 1.

Biomarkers serve as indicators of disease progression or therapeutic response of an medical intervention, and means for enabling a reliable and sensitive biomarker detection are therefore vital in clinical settings. Most biosensor assays require high-affinity interactions in combination with an enzyme or fluorescent tag to enable detection and frequently employ extensive washing procedures prior to signal readout. Attempts to overcome this limitation by using natural biological partners tend to be demanding, because their very low affinity is frequently not compatible with the need of reaching low limits of detection (LODs), especially for circulating biomarkers that possess short half-lives. To address these challenges, we developed a label-free surface plasmon resonance (SPR) platform for the detection of neuregulin 1 (NRG1) using ErbB4-modified liposomes offering both signal amplification and affinity enhancement via functional multivalent interactions. Through the functional avidity interaction between NRG1 and ErbB4, an LOD of 3.5 picomolar was reached, which is about 60-fold higher than traditional SPR and miniaturized immunoassays. The biosensor displays also an 8-fold higher sensitivity when compared with a single-molecule immunoassay employing the natural binding partner rather than a high-affinity antibody as one of the interaction partners. In fact, the liposome-induced avidity between NRG1 and ErbB4 offered an LOD that was comparable to that obtained using a high-affinity antibody and enabled detection of NRG1 in plasma with a LOD of 36 pM. Employing the liposome-enhanced platform in conjunction with a low-affinity biomarker receptor thus enables the assessment of the functional state of the biomarker at competitive LODs and eliminates the need for high-affinity antibodies.

Targeting the NRG1/HER3 pathway in tumor cells and cancer-associated fibroblasts with an anti-neuregulin 1 antibody inhibits tumor growth in pre-clinical models of pancreatic cancer.

Neuregulin 1 (NRG1), a ligand for HER3 and HER4 receptors, is secreted by both pancreatic tumor cells (PC) and cancer-associated fibroblasts (CAFs), the latter representing the most abundant compound of pancreatic stroma. This desmoplastic stroma contributes to Pancreatic Ductal Adenocarcinoma (PDAC) aggressiveness and therapeutic failure by promoting tumor progression, invasion and resistance to chemotherapies. In the present work, we aimed at disrupting the complex crosstalk between PC and CAF in order to prevent tumor cell proliferation. To do so, we demonstrated the promising tumor growth inhibitory effect of the 7E3, an original antibody directed to NRG1. This antibody promotes antibody dependent cellular cytotoxicity in NRG1-positive PC and CAFs and inhibits NRG1-associated signaling pathway induction, by blocking NRG1-mediated HER3 activation. Moreover, 7E3 inhibits migration and growth of pancreatic cancer cells co-cultured with CAFs, both in vitro and in vivo using orthotopic pancreatic tumor xenografts. Our preclinical results demonstrate that the anti-NRG1 antibody 7E3 could represent a promising approach to target pancreatic stroma and cancer cells, thereby providing novel therapeutic options for PDAC.

Antibody-mediated stabilization of NRG1 induces behavioral and electrophysiological alterations in adult mice.

Neuregulin 1 (NRG1) is required for development of the central and peripheral nervous system and regulates neurotransmission in the adult. NRG1 and the gene encoding its receptor, ERBB4, are risk genes for schizophrenia, although how alterations in these genes disrupt their function has not been fully established. Studies of knockout and transgenic mice have yielded conflicting results, with both gain and loss of function resulting in similar behavioral and electrophysiological phenotypes. Here, we used high affinity antibodies to NRG1 and ErbB4 to perturb the function of the endogenous proteins in adult mice. Treatment with NRG1 antibodies that block receptor binding caused behavioral alterations associated with schizophrenia, including, hyper-locomotion and impaired pre-pulse inhibition of startle (PPI). Electrophysiological analysis of brain slices from anti-NRG1 treated mice revealed reduced synaptic transmission and enhanced paired-pulse facilitation. In contrast, mice treated with more potent ErbB4 function blocking antibodies did not display behavioral alterations, suggesting a receptor independent mechanism of the anti-NRG1-induced phenotypes. We demonstrate that anti-NRG1 causes accumulation of the full-length transmembrane protein and increases phospho-cofilin levels, which has previously been linked to impaired synaptic transmission, indicating enhancement of non-canonical NRG1 signaling could mediate the CNS effects.

A TLR/AKT/FoxO3 immune tolerance-like pathway disrupts the repair capacity of oligodendrocyte progenitors.

Cerebral white matter injury (WMI) persistently disrupts myelin regeneration by oligodendrocyte progenitor cells (OPCs). We identified a specific bioactive hyaluronan fragment (bHAf) that downregulates myelin gene expression and chronically blocks OPC maturation and myelination via a tolerance-like mechanism that dysregulates pro-myelination signaling via AKT. Desensitization of AKT occurs via TLR4 but not TLR2 or CD44. OPC differentiation was selectively blocked by bHAf in a maturation-dependent fashion at the late OPC (preOL) stage by a noncanonical TLR4/TRIF pathway that induced persistent activation of the FoxO3 transcription factor downstream of AKT. Activated FoxO3 selectively localized to oligodendrocyte lineage cells in white matter lesions from human preterm neonates and adults with multiple sclerosis. FoxO3 constraint of OPC maturation was bHAf dependent, and involved interactions at the FoxO3 and MBP promoters with the chromatin remodeling factor Brg1 and the transcription factor Olig2, which regulate OPC differentiation. WMI has adapted an immune tolerance-like mechanism whereby persistent engagement of TLR4 by bHAf promotes an OPC niche at the expense of myelination by engaging a FoxO3 signaling pathway that chronically constrains OPC differentiation.

Neuregulin 1 Allosterically Enhances the Antitumor Effects of the Noncompeting Anti-HER3 Antibody 9F7-F11 by Increasing Its Binding to HER3.

Exploratory clinical trials using therapeutic anti-HER3 antibodies strongly suggest that neuregulin (NRG1; HER3 ligand) expression at tumor sites is a predictive biomarker of anti-HER3 antibody efficacy in cancer. We hypothesized that in NRG1-expressing tumors, where the ligand is present before antibody treatment, anti-HER3 antibodies that do not compete with NRG1 for receptor binding have a higher receptor-neutralizing action than antibodies competing with the ligand for binding to HER3. Using time-resolved-fluorescence energy transfer (TR-FRET), we demonstrated that in the presence of recombinant NRG1, binding of 9F7-F11 (a nonligand-competing anti-HER3 antibody) to HER3 is increased, whereas that of ligand-competing anti-HER3 antibodies (H4B-121, U3-1287, Ab#6, Mab205.10.2, and MOR09825) is decreased. Moreover, 9F7-F11 showed higher efficacy than antibodies that compete with the ligand for binding to HER3. Specifically, 9F7-F11 inhibition of cell proliferation and of HER3/AKT/ERK1/2 phosphorylation as well as 9F7-F11-dependent cell-mediated cytotoxicity were higher in cancer cells preincubated with recombinant NRG1 compared with cells directly exposed to the anti-HER3 antibody. This translated in vivo into enhanced growth inhibition of NRG1-expressing BxPC3 pancreatic, A549 lung, and HCC-1806 breast cell tumor xenografts in mice treated with 9F7-F11 compared with H4B-121. Conversely, both antibodies had similar antitumor effect in NRG1-negative HPAC pancreatic carcinoma cells. In conclusion, the allosteric modulator 9F7-F11 shows increased anticancer effectiveness in the presence of NRG1 and thus represents a novel treatment strategy for NRG1-addicted tumors. Mol Cancer Ther; 16(7); 1312-23. ©2017 AACR.

Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV.

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3-43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (KD = 220 pM) and HER3-expressing tumor cells with EC50 values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3-43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3-43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells.

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix.

Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor.

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFbeta isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFbeta1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

IFN-γ Rα is a key determinant of CD8+ T cell-mediated tumor elimination or tumor escape and relapse in FVB mouse.

During the past decade, the dual function of the immune system in tumor inhibition and tumor progression has become appreciated. We have previously reported that neu-specific T cells can induce rejection of neu positive mouse mammary carcinoma (MMC) and also facilitate tumor relapse by inducing neu antigen loss and epithelial to mesenchymal transition (EMT). Here, we sought to determine the mechanism by which CD8+ T cells either eliminate the tumor, or maintain tumor cells in a dormant state and eventually facilitate tumor relapse. We show that tumor cells that express high levels of IFN-γ Rα are eliminated by CD8+ T cells. In contrast, tumor cells that express low levels of IFN-γ Rα do not die but remain dormant and quiescent in the presence of IFN-γ producing CD8+ T cells until they hide themselves from the adaptive immune system by losing the tumor antigen, neu. Relapsed tumor cells show CD44+CD24- phenotype with higher rates of tumorigenesis, in vivo. Acquisition of CD44+CD24- phenotype in relapsed tumors was not solely due to Darwinian selection. Our data suggest that tumor cells control the outcome of tumor immune surveillance through modulation of the expression of    IFN-γ Rα.

Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor / Producción y evaluación de anticuerpos de gallinas contra un péptido sintético del factor de crecimiento glial

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFb isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFb1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

Las Neuregulinas (NRG) son proteínas que pertenecen a la familia de los factores de crecimiento epidermal. Se ha demostrado que estos factores son esenciales para el desarrollo y mantenimiento de la funcionalidad del sistema nervioso. Debido a la dificultad para purificar estas proteínas y la falta de especificidad de los anticuerpos disponibles comercialmente, el objetivo de este trabajo fue producir anticuerpos contra un péptido sintético capaz de detectar e identificar una isoforma de la Neuregulina (GGFb). Para lograr este objetivo, se desarrollaron anticuerpos en gallinas (IgY) contra un péptido sintético diseñado a partir de la secuencia aminoacídica de la región extracelular de GGFb, utilizando programas de predicción de epítopes. Los resultados demuestran que el péptido seleccionado fue immunogénico debido a que estimuló una respuesta inmune específica tipo B en el modelo utilizado. Estos anticuerpos fueron también capaces de reconocer una proteína recombinante e isoformas de GGF presentes en diferentes muestras biológicas. Nuestros resultados demuestran el potencial valor de las inmunoglobulinas Y (IgY) contra péptidos sintéticos como una herramienta de aplicación para la investigación en neurociencia.

In vivo and in vitro genetic evidence of involvement of neuregulin 1 in immune system dysregulation.

Neuregulin 1 (NRG1) has been implicated in several disorders including breast cancer, multiple sclerosis, and schizophrenia. Also, recent evidence suggests that NRG1 may play a role in regulation of inflammation and immune system response. We therefore hypothesized that a schizophrenia-associated missense mutation (valine to leucine) we identified within the transmembrane region of NRG1 would also be linked to immune dysregulation. We used plasma samples from families carrying the mutation to measure levels of antibodies to 41 autoimmune markers and six cytokines (IL-1b, IL-6, IL-10, IL-8, IL-12p70, and TNF-α) and used these levels as quantitative traits to evaluate association with the NRG1 mutation, using FBAT. Next, we used Epstein-Barr virus-transformed B cells from heterozygous mutation carriers and wild-type individuals to evaluate protein and mRNA cytokine expression in vitro using quantitative PCR and ELISA assays. In vivo, increased levels of 25 autoimmune markers as well as elevated levels of cytokines were significantly associated with the NRG1 mutation. In vitro, we observed a significant increase in protein secretion levels of IL-6, TNF-α, and IL-8 in mutation carriers compared with controls. At the mRNA level, we observed a significant increase in IL-6 expression, while IL-4 levels appeared to be down-regulated in heterozygous individuals compared with wild-type controls. This is the first report of association of a NRG1 mutation with immune dysregulation. This study could contribute towards understanding the role of NRG1 in the pathogenesis of schizophrenia and other disorders in which inflammation plays an important role.

Preventive effects of heregulin-beta1 on macrophage foam cell formation and atherosclerosis.

RATIONALE: Human heregulins, neuregulin-1 type I polypeptides that activate proliferation, differentiation, and survival of glial cells, neurons, and myocytes, are expressed in macrophage foam cells within human coronary atherosclerotic lesions. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor class A (SR-A), acyl-coenzyme A:cholesterol acyltransferase (ACAT)1, and ATP-binding cassette transporter (ABC)A1. OBJECTIVE: The present study clarified the roles of heregulins in macrophage foam cell formation and atherosclerosis. METHODS AND RESULTS: Plasma heregulin-beta(1) levels were significantly decreased in 31 patients with acute coronary syndrome and 33 patients with effort angina pectoris compared with 34 patients with mild hypertension and 40 healthy volunteers (1.3+/-0.3, 2.0+/-0.4 versus 7.6+/-1.4, 8.2+/-1.2 ng/mL; P<0.01). Among all patients with acute coronary syndrome and effort angina pectoris, plasma heregulin-beta(1) levels were further decreased in accordance with the severity of coronary artery lesions. Expression of heregulin-beta(1) was observed at trace levels in intracoronary atherothrombosis obtained by aspiration thrombectomy from acute coronary syndrome patients. Heregulin-beta(1), but not heregulin-alpha, significantly reduced acetylated low-density lipoprotein-induced cholesterol ester accumulation in primary cultured human monocyte-derived macrophages by reducing SR-A and ACAT1 expression and by increasing ABCA1 expression at both mRNA and protein levels. Heregulin-beta(1) significantly decreased endocytic uptake of [(125)I]acetylated low-density lipoprotein and ACAT activity, and increased cholesterol efflux to apolipoprotein (Apo)A-I from human macrophages. Chronic infusion of heregulin-beta(1) into ApoE(-/-) mice significantly suppressed the development of atherosclerotic lesions. CONCLUSIONS: This study provided the first evidence that heregulin-beta(1) inhibits atherogenesis and suppresses macrophage foam cell formation via SR-A and ACAT1 downregulation and ABCA1 upregulation.

Efficacy of a genetically engineered Candida albicans tet-NRG1 strain as an experimental live attenuated vaccine against hematogenously disseminated candidiasis.

We report on the efficacy of the genetically engineered Candida albicans tet-NRG1 strain as an experimental live, attenuated vaccine against disseminated candidiasis in both immunocompetent and immunodeficient mice mostly dependent on T-cell immunity. This experimental vaccination model may represent an important tool to unravel the mechanisms of protective immunity during candidiasis.

Elevated neuregulin-1 and ErbB4 protein in the prefrontal cortex of schizophrenic patients.

Neuregulin-1 (NRG1) and its receptor, ErbB4, have been implicated in schizophrenia at both gene and transcript levels. The present investigation compared NRG1 and ErbB4 protein levels in prefrontal cortical (PFC) cytoplasmic and nuclear fractions among normal, schizophrenic, bipolar and major depressed subjects from the Stanley Consortium. We used immunoblotting procedures to examine potential NRG1 and ErbB4 immunoreactive bands, but specifically quantified NRG1 immunoreactive signals at 42, 48 and 53 kDa and ErbB4 immunoreactive signals at 21, 55, 60 and 180 kDa. PFC cytoplasmic 53 kDa NRG1 protein levels were significantly increased (approximately 20%) in schizophrenic patients relative to each of the other subject groups. We also detected diagnostic effects on PFC cytoplasmic full-length (180 kDa) ErbB4 protein levels, and post hoc tests revealed that these quantities were significantly increased (approximately 30%) in schizophrenic patients relative to normal and to depressed subjects. In addition, we examined the levels of potential ErbB4 cleavage products at 21, 55 and 60 kDa relative to those of full-length ErbB4 in the PFC fractions. We detected trends for diagnostic effects on PFC cytoplasmic 21 kDa/180 kDa and 55 kDa/180 kDa ratios, and post hoc tests revealed that these ratios were significantly reduced in schizophrenic patients relative to normal individuals. Our investigation suggests that schizophrenia-associated NRG1 and ErbB4 mRNA elevations also occur at the protein level and may be specific to schizophrenia. We hypothesize that ErbB4 proteolytic processing may also be altered in schizophrenia, yielding altered ratios of functionally distinct forms of ErbB4.

H-RAS V12-induced radioresistance in HCT116 colon carcinoma cells is heregulin dependent.

The abilities of mutated active K-RAS and H-RAS proteins, in an isogenic human carcinoma cell system, to modulate the activity of signaling pathways following exposure to ionizing radiation is unknown. Loss of K-RAS D13 expression in HCT116 colorectal carcinoma cells blunted basal extracellular signal-regulated kinase 1/2 (ERK1/2), AKT, and c-Jun NH2-terminal kinase 1/2 activity. Deletion of the allele to express K-RAS D13 also enhanced expression of ERBB1, ERBB3, and heregulin but nearly abolished radiation-induced activation of all signaling pathways. Expression of H-RAS V12 in HCT116 cells lacking an activated RAS molecule (H-RAS V12 cells) restored basal ERK1/2 and AKT activity to that observed in parental cells but did not restore or alter basal c-jun NH2-terminal kinase 1/2 activity. In parental cells, radiation caused stronger ERK1/2 pathway activation compared with that of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which correlated with constitutive translocation of Raf-1 into the plasma membrane of parental cells. Inhibition of mitogen-activated protein kinase/ERK1/2, but not PI3K, radiosensitized parental cells. In H-RAS V12 cells, radiation caused stronger PI3K/AKT pathway activation compared with that of the ERK1/2 pathway, which correlated with H-RAS V12-dependent translocation of PI3K into the plasma membrane. Inhibition of PI3K, but not mitogen-activated protein kinase/ERK1/2, radiosensitized H-RAS V12 cells. Radiation-induced activation of the PI3K/AKT pathway in H-RAS V12 cells 2 to 24 hours after exposure was dependent on heregulin-stimulated ERBB3 association with membrane-localized PI3K. Neutralization of heregulin function abolished radiation-induced AKT activation and reverted the radiosensitivity of H-RAS V12 cells to those levels found in cells lacking expression of any active RAS protein. These findings show that H-RAS V12 and K-RAS D13 differentially regulate radiation-induced signaling pathway function. In HCT116 cells expressing H-RAS V12, PI3K-dependent radioresistance is mediated by both H-RAS-dependent translocation of PI3K into the plasma membrane and heregulin-induced activation of membrane-localized PI3K via ERBB3.

A real-time quantitative PCR comparative study between rat optic and sciatic nerves: determination of neuregulin-1 mRNA levels.

Injured axons from peripheral nervous system (PNS) possess the ability to regenerate. In contrast, regeneration of injured axons does not occur in the central nervous system (CNS) or occurs to a limited extent. Previous works have shown that rat sciatic nerve conditioned medium (CM) produced PC12 cells neuronal-like differentiation and neurite outgrowth. In the present work, we compared the expression of neuregulin-1s (NRG-1s) from rat sciatic and optic nerves as members of the PNS and CNS, respectively. Sciatic nerve CM showed a higher neurotrophic activity on PC12 cells than rat optic nerve CM. RT-PCR analysis verified the presence of all three types of NRG-1 mRNAs and their receptors in both types of nerves. Real-time quantitative PCR (QPCR) assays showed that the relative expression levels of all three types of NRG-1 mRNAs were higher in optic nerves than in sciatic nerves. Eleven-day cultured optic nerves showed an increased in NDF and SMDF when compared to freshly isolated optic nerves, whereas GGF decreased. However, 11-day-cultured sciatic nerves only showed an increase in SMDF mRNA. Western blots corroborated the differences in NRG-1 expression profile for both types of nerves and their CMs. Incubation of both CMs with the anti-pan-NRG-1 antibody showed that the neurotrophic activity of the optic nerve CM increased, whereas the sciatic nerve CM remained unchanged. These results indicated that different NRG-1 levels are expressed upon nerve degeneration and the balance between those levels and other neurotrophic factors could have an important role on nerve regeneration.

Autocrine heregulin generates growth factor independence and blocks apoptosis in colon cancer cells.

The aim of this study was to determine whether constitutive ErbB2 activation controls growth and apoptosis in colon cancer cells. Growth arrested GEO cells showed constitutive activation of ErbB2 in the absence of exogenous growth factors or serum supplementation. Higher levels of heregulin and ErbB2 activation were observed in the growth-arrested state and cell cycle re-entry was independent of exogenous growth factors. Blockade of ErbB2 activation by heregulin neutralizing antibodies and by AG879 resulted in prevention of cell cycle re-entry. This indicated that autocrine heregulin activity was responsible for growth factor independence and for cell cycle re-entry. Activation of ErbB2 was the result of heregulin mediated interaction with ErbB3 and generated downstream activation of the ERK and the PI3K/AKT pathways. Heregulin neutralizing antibody treatment of growth arrested GEO cells also generated apoptosis as reflected by PARP cleavage and DNA fragmentation indicating a cell survival signal was also induced by the constitutively activated ErbB2. The activation of AKT but not the MAPK pathway was responsible for cell survival in these cells.

Regulation of elongation factor-1alpha expression by growth factors and anti-receptor blocking antibodies.

The epidermal growth factor (EGF) family and its receptors regulate normal and cancerous epithelial cell proliferation, a process that could be suppressed by anti-receptor blocking antibodies. Polypeptide elongation factor-1alpha (EF-1alpha) is a multifunctional protein whose levels are positively correlated with the proliferative state of cells. To identify genes, whose expression may be modulated by anti-receptor blocking antibodies, we performed a differential display screening and isolated differentially expressed cDNAs. Isolates from one clone were 100% identical to human EF-1alpha. Both EGF and heregulin-beta1 (HRG) induced EF-1alpha promoter activity and mRNA and protein expression. Growth factor-mediated EF-1alpha expression was effectively blocked by pretreatment with humanized anti-EGF receptor antibody C225 or anti-human epidermal growth factor receptor-2 (HER2) antibody herceptin. Mutants and pharmacological inhibitors of p38(MAPK) and MEK, but not phosphatidylinositol 3-kinase, suppressed both constitutive and HRG-induced stimulation of EF-1alpha promoter activity in MCF-7 cells. Deletion analysis of the promoter suggested the requirement of the -393 to -204 region for growth factor-mediated transcription of EF-1alpha. Fine mapping and point mutation studies revealed a role of the SP1 site in the observed HRG-mediated regulation of the EF-1alpha promoter. In addition, we also provide new evidence to suggest that HRG stimulation of the EF-1alpha promoter involves increased physical interactions with acetylated histone H3 and histone H4. These results suggest that regulation of EF-1alpha expression by extracellular signals that function through human EGF receptor family members that are widely deregulated in human cancers and that growth factor regulation of EF-1alpha expression involve histone acetylation.