Neuregulin Research Articles

MicroRNA Expression in the Infarcted Heart Following Neonatal Cardiovascular Progenitor Cell Transplantation in a Sheep Model of Stem Cell–Based Repair

Myocardial infarctions affect approximately 735,000 people annually in the United States and have a substantial impact on quality of life. Neonates have an enhanced capability of repairing cardiovascular damage, while adults do not. The mechanistic basis for this age-dependent difference in regenerative capacity remains unknown. Recent studies have shown that microRNAs (miRNAs) play a significant role in regulating the regenerative ability of cardiovascular cells. This report defines the alterations in miRNA expression within the cardiovascular repair zone of infarcted sheep hearts following intracardiac injection of neonatal islet-1+ cardiovascular progenitor cells. Sheep were infarcted via left anterior descending coronary artery ligation. After 3 to 4 weeks of infarction, sheep neonatal islet-1+ cardiovascular progenitor cells were injected into the infarcted area for repair. Cell-treated sheep were euthanized 2 months following cell injection, and their hearts were harvested for the analysis of miRNA and gene expression within the cardiovascular repair zone. Ten miRNAs were differentially regulated in vivo, including miR-99, miR-100, miR-302a, miR-208a, miR-665, miR-1, miR-499a, miR-34a, miR-133a, and miR-199a. These miRNAs promote stemness, cell division, and survival. Several signaling pathways are regulated by these miRNAs, including Hippo, Wnt, and Erythroblastic Leukemia Viral Oncogene B (ERBB). Transcripts encoding Wnt, ERBB, and Neuregulin 1 (NRG1) were elevated in vivo in the infarct repair zone. Wnt5a signaling and ERBB/NRG1 transcripts contribute to activation of Yes-Associated Protein 1. MiRNAs that impact proliferation, cell survival, and signaling pathways that promote regeneration were induced during cardiovascular repair in the sheep model. This information can be used to design new approaches for the optimization of miRNA-based treatments for the heart.

Cytokines in Schizophrenia: Predisposing Factors and Inflammatory Responses

Schizophrenia is a multifactorial disease that may be triggered by environmental disorders, mental stress, or physical illness in a person who is genetically predisposed to it. Recent research has extremely evaluated the role of inflammatory cytokines in schizophrenia. The current study attempted to find any association between cytokines and schizophrenia. This narrative study was conducted through a literature review in Scopus, PubMed, and Web of Science databases using keywords related to schizophrenia and cytokines without any time limitation. This review focused on discussing the relationship between various symptoms of disease and cytokine levels. Environmental factors such as embryonical infections were found to be associated with alternations in mothers’ blood cytokines and further effects on schizophrenia probability in children. Heritable factors including disrupted-in-schizophrenia 1 (DISC1) and neuregulin 1 (NRG1) genes were shown to be linked with immunological responses, as well as Toll-like receptors or AKT1/p13k activation and following alternations in the cytokine profile. Based on the findings, the cytokines and inflammatory responses of the body were reported to be involved in many psychological disorders and schizophrenia. Various interleukins (ILs), as well as IL-6, IL-8, and IL-2R are known to be associated with the severity of schizophrenia symptoms. Although various data exist regarding different cytokines and their association with schizophrenia, no study has so far formulated research fields on specific types of cytokines that have the potential to be further studied for therapeutic hope.

A new strategy for stem cells therapy for erectile dysfunction: Adipose-derived stem cells transfect Neuregulin-1 gene through superparamagnetic iron oxide nanoparticles.

PurposeOur previous studies showed that nanotechnology improves derived adipose-derived stem cells (ADSCs) therapy for erectile dysfunction (ED). In this study, the Neuregulin-1(NRG1) gene was transfected into ADSCs with superparamagnetic iron oxide nanoparticles (SPION) further to improve the therapeutic effect of ADSCs on ED.Materials and MethodsADSCs were isolated from epididymal adipose tissue of Sprague–Dawley rats. The optimal concentration of PEI-SPION (SPION modified with polyethyleneimine) was selected to construct the gene complex. After electrostatic binding of PEI-SPION and DNA, a PEI layer was wrapped to make the PEI-SPION-NRG1-PEI gene transfection complex. Different groups were set up for transfection tests. Lipo2000 transfection reagent was used as the control. PEI-SPION-NRG1-PEI in the experimental group was transfected under an external magnetic field.ResultsWhen the concentration of PEI-SPION was 10 µg/mL, it had little cytotoxicity, and cell activity was not significantly affected. PEI-SPION-NRG1-PEI forms positively charged nanocomposites with a particle size of 72.6±14.9 nm when N/P ≥8. The PEI-SPION-NRG1-PEI gene complex can significantly improve the transfection efficiency of ADSCs, reaching 26.74%±4.62%, under the action of the external magnetic field. PCR and Western blot showed that the expression level of the NRG1 gene increased significantly, which proved that the transfection was effective.ConclusionsPEI-SPION can be used as a vector for NRG1 gene transfection into ADSCs. PEI-SPION-NRG1-PEI packaging has the highest transfection efficiency under the external magnetic field than the other groups. These findings may provide a new strategy for ADSCs therapy for ED.

Epigenetic Biomarkers as Diagnostic Tools for Neurodegenerative Disorders.

Epigenetics is the study of heritable changes in gene expression that occur without alterations to the DNA sequence, linking the genome to its surroundings. The accumulation of epigenetic alterations over the lifespan may contribute to neurodegeneration. The aim of the present study was to identify epigenetic biomarkers for improving diagnostic efficacy in patients with neurodegenerative diseases. We analyzed global DNA methylation, chromatin remodeling/histone modifications, sirtuin (SIRT) expression and activity, and the expression of several important neurodegeneration-related genes. DNA methylation, SIRT expression and activity and neuregulin 1 (NRG1), microtubule-associated protein tau (MAPT) and brain-derived neurotrophic factor (BDNF) expression were reduced in buffy coat samples from patients with neurodegenerative disorders. Our data suggest that these epigenetic biomarkers may be useful in clinical practical for the diagnosis, surveillance, and prognosis of disease activity in patients with neurodegenerative diseases.

Conditional ERK3 overexpression cooperates with PTEN deletion to promote lung adenocarcinoma formation in mice.

ERK3, officially known as mitogen‐activated protein kinase 6 (MAPK6), is a poorly studied mitogen‐activated protein kinase (MAPK). Recent studies have revealed the upregulation of ERK3 expression in cancer and suggest an important role for ERK3 in promoting cancer cell growth and invasion in some cancers, in particular lung cancer. However, it is unknown whether ERK3 plays a role in spontaneous tumorigenesis in vivo. To determine the role of ERK3 in lung tumorigenesis, we created a conditional ERK3 transgenic mouse line in which ERK3 transgene expression is controlled by Cre recombinase. By crossing these transgenic mice with a mouse line harboring a lung tissue–specific Cre recombinase transgene driven by a club cell secretory protein gene promoter (CCSP‐iCre), we have found that conditional ERK3 overexpression cooperates with phosphatase and tensin homolog (PTEN) deletion to induce the formation of lung adenocarcinomas (LUADs). Mechanistically, ERK3 overexpression stimulates activating phosphorylations of erb‐b2 receptor tyrosine kinases 2 and 3 (ERBB2 and ERBB3) by upregulating Sp1 transcription factor (SP1)–mediated gene transcription of neuregulin 1 (NRG1), a potent ligand for ERBB2/ERBB3. Our study has revealed a bona fide tumor‐promoting role for ERK3 using genetically engineered mouse models. Together with previous findings showing the roles of ERK3 in cultured cells and in a xenograft lung tumor model, our findings corroborate that ERK3 acts as an oncoprotein in promoting LUAD development and progression.

Hepatic Proteomics Analysis of Nonalcoholic Fatty Liver Disease Obese Rat Model After Short- and Long-Term Soy Protein Isolate Feeding.

To identify possible mechanisms involved in the development and progression of nonalcoholic fatty liver disease (NAFLD), we conducted shotgun proteomics analysis on liver of obese Zucker rats fed either casein (CAS) or soy protein isolate (SPI) for 8 and 16 weeks. Rats (7 weeks old, n = 8-9/group) were randomly assigned to either a CAS-based or an SPI-based diet. Rats were killed after 8 or 16 weeks of feeding and livers were stored at -80°C. Ingenuity Pathway Analysis (IPA) software was used to facilitate interpretation of proteomics data. Predictions of activation or inhibition of molecules in the data were made based on activation z-score and P value of overlap (P < .05). Activation z-scores ≥2.0 indicate that a molecule is predicted to be activated, whereas activation z-scores of less than or equal to -2.0 indicate that a target molecule is predicted to be inhibited. Upstream regulator analysis with IPA revealed Neuregulin 1 (NRG1) to be the top activated protein in (z-score = 2.48, P < .05), and MKNK1 as the top inhibited protein (z-score = -2.83, P < .05) in SPI diet compared with CAS diet after both 8 and 16 weeks of SPI feeding. Regulator effects analysis also predicted that some proteins would be participating, directly or indirectly, in the inhibition of immune response functions (such as leukocyte migration) and lipid metabolism (such as synthesis of lipids) in SPI-fed rats relative to CAS-fed rats. Our results suggest that SPI diet modifies the expression of proteins that could be involved in the reduction of NAFLD.

Abstract P067: VRN10s, a series of HER2 inhibitors to overcome NRG-mediated drug resistance and acquired-drug resistant HER2 mutations

Abstract The overexpression of Human epidermal growth factor receptor 3 (HER3) and increased Neuregulin (NRG) production are well known resistance mechanisms to Human epidermal growth factor receptor 2 (HER2) kinase inhibitors. FDA-approved HER2 kinase inhibitors show significantly decreased activities in the presence of NRG, which induces HER2-HER3 heterodimer and activates HER3. VRN10s, HER2 kinase inhibitors, maintained potency to inhibit proliferation of HER2-positive breast cancer cell lines even in the presence NRG1. Western blot confirmed that VRN10s inhibited phosphorylation of HER2 and HER3 as well as downstream signaling, such as AKT and ERK pathways. Emergence of acquired resistance mutations against tyrosine kinase inhibitors (TKI) are also nearly inevitable, but less is known about the acquired drug resistance mutations of the HER2 kinase domain. To predict the drug resistance mutations against two representative HER2 TKIs, Tucatinib (non-covalent) and Neratinib (covalent), we performed an ENU mutagenesis assay and identified HER2 mutant sequences from clonal cells survived from Tucatinib and Neratinib treatment in Ba/F3 HER2 WT cells. There were 9 types of Tucatinib-resistant mutations. The most common Tucatinib-resistant mutation type was T862A/S, which was found in 5 out of total 15 clones. All types of mutations showed 6~70 folds resistance for Tucatinib compared to WT. These mutant types were also resistant against another non-covalent HER2 TKI, Lapatinib. In case of Neratinib, HER2 S963P was derived from two different clones, showing 4~8 folds resistance against Neratinib compared to WT. However, a series of VRN10 was relatively sensitive toward all Tucatinib or Neratinib resistant mutants. Based on our in vitro assay and ENU mutagenesis studies, VRN10s can be a new option to overcome HER2 TKI drug resistance derived by HER3 activation or HER2 mutagenesis. Citation Format: Chan Mi Park, Jihye Yoo, Mingyeong Son, Hong-ryul Jung, Jin-Hee Park, Jeongbum Son, Eunhwa Ko, Sunghwan Kim. VRN10s, a series of HER2 inhibitors to overcome NRG-mediated drug resistance and acquired-drug resistant HER2 mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P067.

NRG1/ErbB4 signaling and its role on the development and progression of Alzheimer's disease

AbstractBackgroundAlzheimer’s disease (AD) is a complex disease; various etiopathogenic mechanisms, like genetics and cognitive reserve, affect its development and progression across the continuum. Neuregulin‐1 (NRG‐1) /ErbB4 signaling has been recently studied regarding its possible role in the development of AD. To our knowledge, the schizophrenia (SZ) risk SNPs rs6994992 (NRG‐1) and rs839523 (ErbB4) have never been explored in association to AD, but the risk alleles have been associated with structural, functional and cognitive alterations in SZ and healthy individuals. Some of these alterations are similar to those experienced by AD patients, over 40% of which evolve to experience psychotic symptoms. This subset of patients experience a more rapid cognitive decline, greater functional impairment, etc., which contribute to a greater level of distress for family and caregivers.MethodThe sample consisted of 584 Spanish individuals: 161 were diagnosed with mild cognitive impairment (MCI; age = 76.66±5.37). The remaining 423 were healthy controls (HC; age = 65.88±8.80).Participants underwent a neuropsychological evaluation, a MEG scan, a magnetic resonance imaging scan, and were genotyped for the rs6994992 and rs839523 SNPs.ResultPreliminary results show a significant interaction between the NRG1 risk allele and the diagnosis in the MMSE score (figure 1); the risk allele had no effect in HC, but in MCI those with the risk allele had a significantly worse performance. Linear models were performed for the prediction of the MMSE score, including either the NRG1 or ErbB4 SNP, the diagnosis, cognitive reserve and age. Significant interactions with the SNPs were found for both, including a three‐way interaction between the diagnosis, cognitive reserve.ConclusionThese preliminary findings could indicate that the SZ risk alleles have a negative influence in the AD continuum over cognition and, potentially, over structural and functional parameters. This effect could be modulated by cognitive activity across the lifespan.

The role of the KIBRA and APOE genes in developing spatial abilities in humans.

In the contemporary high-tech society, spatial abilities predict individual life and professional success, especially in the STEM (Science, Technology, Engineering, and Mathematics) disciplines. According to neurobiological hypotheses, individual differences in cognitive abilities may be attributed to the functioning of genes involved in the regulation of neurogenesis and synaptic plasticity. In addition, genome-wide association studies identified rs17070145 located in the KIBRA gene, which was associated with individual differences in episodic memory. Considering a significant role of genetic and environmental components in cognitive functioning, the present study aimed to estimate the main effect of NGF (rs6330), NRXN1 (rs1045881, rs4971648), KIBRA (rs17070145), NRG1 (rs6994992), BDNF (rs6265), GRIN2B (rs3764030), APOE (rs7412, rs429358), and SNAP25 (rs363050) gene polymorphisms and to assess the effect of gene-environment interactions on individual differences in spatial ability in individuals without cognitive decline aged 18–25 years (N = 1011, 80 % women). Spatial abilities were measured using a battery of cognitive tests including the assessment of “3D shape rotation” (mental rotation). Multiple regression analysis, which was carried out in the total sample controlling for sex, ethnicity and the presence of the “risk” APOE ε4 allele, demonstrated the association of the rs17070145 Т-allele in the KIBRA gene with enhanced spatial ability (β = 1.32; pFDR = 0.037) compared to carriers of the rs17070145 CC-genotype. The analysis of gene-environment interactions revealed that nicotine smoking (β = 3.74; p = 0.010) and urban/rural residency in childhood (β = –6.94; p = 0.0002) modulated the association of KIBRA rs17070145 and АРОЕ (rs7412, rs429358) gene variants with individual differences in mental rotation, respectively. The data obtained confirm the effect of the KIBRA rs17070145 Т-allele on improved cognitive functioning and for the first time evidence the association of the mentioned genetic variant with spatial abilities in humans. A “protective” effect of the APOE ε2 allele on enhanced cognitive functioning is observed only under certain conditions related to childhood rearing.

Neuregulin 1 (NRG1) fusion-positive high-grade spindle cell sarcoma: A distinct group of soft tissue tumors with metastatic potential.

Neuregulin 1 (NRG1) is an epidermal growth factor (EGF)-like ligand that activates receptor tyrosine kinases of the ErbB family of receptors. NRG1 gene fusions, which are rare (<1%) but recurrent events in solid tumors, are an emerging oncogenic driver that is potentially actionable using ErbB-targeted tyrosine kinase inhibitors. Largely characterized only in carcinomas, we describe three cases of NRG1-rearranged sarcomas. The patients were all female, aged 32-47 years old. Two cases were deep-seated tumors in the lower extremities (right thigh and calf); one case presented as a uterine mass. The tumors measured 9-11.5 cm in the greatest dimensions. Histologically, all three tumors were high-grade spindle cell sarcomas composed of monomorphic spindle cells arranged in interlacing fascicles. The tumor cells were set in the loose collagenous stroma with branching, curvilinear thin-walled vasculature in the background. Cytologically, the neoplastic cells displayed ovoid to fusiform nuclei with finely stippled chromatin, inconspicuous nucleoli, scant to moderate clear to eosinophilic cytoplasm, occasional cytoplasmic vacuoles, and elongated cytoplasmic processes. Mitotic activity was elevated (> 20/10 high power fields) and tumor necrosis was present. None of the tumors expressed lineage-specific immunophenotypical markers. Targeted RNA-sequencing uncovered gene fusions involving NRG1 and the 5' untranslated regions of PPHLN1, HMBOX1, or MTUS1. In all cases, the C-terminal EGF-like domain of NRG1 was preserved in the predicted chimeric protein product. All three patients developed metastatic disease within 2 years from initial presentation and were alive with disease at last follow-up (mean follow-up period=19 months). In conclusion, we present the first case series of NRG1-rearranged sarcomas characterized by high-grade fascicular spindle cell morphology, non-specific immunoprofile, and aggressive clinical behavior. Further studies are needed to determine whether this distinct subgroup of spindle cell sarcomas are amenable to targeted therapies.

Neuregulin 1 and ErbB4 Kinase Actively Regulate Sharp Wave Ripples in the Hippocampus

Sharp wave ripples (SW-Rs) in the hippocampus are synchronized bursts of hippocampal pyramidal neurons (PyNs), critical for spatial working memory. However, the molecular underpinnings of SW-Rs remain poorly understood. We show that SW-Rs in hippocampal slices from both male and female mice were suppressed by neuregulin 1 (NRG1), an epidermal growth factor whose expression is enhanced by neuronal activity. Pharmacological inhibition of ErbB4, a receptor tyrosine kinase for NRG1, increases SW-R occurrence rate in hippocampal slices. These results suggest an important role of NRG1-ErbB4 signaling in regulating SW-Rs. To further test this notion, we characterized SW-Rs in freely moving male mice, chemical genetic mutant mice, where ErbB4 can be specifically inhibited by the bulky inhibitor 1NMPP1. Remarkably, SW-R occurrence was increased by 1NMPP1. We found that 1NMPP1 increased the firing rate of PyN neurons, yet disrupted PyN neuron dynamics during SW-R events. Furthermore, 1NMPP1 increased SW-R occurrence during both nonrapid eye movement (NREM) sleep states and wake states with a greater impact on SW-Rs during wake states. In accord, spatial working memory was attenuated in male mice. Together these results indicate that dynamic activity of ErbB4 kinase is critical to SW-Rs and spatial working memory. This study reveals a novel regulatory mechanism of SW-Rs and a novel function of the NRG1-ErbB4 signaling.SIGNIFICANCE STATEMENT Sharp wave ripples (SW-Rs) are a hippocampal event, important for memory functioning. Yet the molecular pathways that regulate SW-Rs remain unclear. Neuregulin 1 (NRG1), previously known to be increased in pyramidal neuron's (PyNs) in an activity dependent manner, signals to its receptor, ErbB4 kinase, that is in important regulator of GABAergic transmission and long-term potentiation in the hippocampus. Our findings demonstrate that SW-Rs are regulated by this signaling pathway in a dynamic manner. Not only so, we show that this signaling pathway is dynamically needed for spatial working memory. These data suggest a molecular signaling pathway, NRG1-ErbB4, that regulates an important network event of the hippocampus, SW-Rs, that underlies memory functioning.

Cre-Activation in ErbB4-Positive Neurons of Floxed Grin1/NMDA Receptor Mice Is Not Associated With Major Behavioral Impairment.

Extensive evidence suggests a dysfunction of the glutamate NMDA receptor (NMDAR) in schizophrenia, a severe psychiatric disorder with putative early neurodevelopmental origins, but clinical onset mainly during late adolescence. On the other hand, pharmacological models using NMDAR antagonists and the clinical manifestation of anti-NMDAR encephalitis indicate that NMDAR blockade/hypofunction can trigger psychosis also at adult stages, without any early developmental dysfunction. Previous genetic models of NMDAR hypofunction restricted to parvalbumin-positive interneurons indicate the necessity of an early postnatal impairment to trigger schizophrenia-like abnormalities, whereas the cellular substrates of NMDAR-mediated psychosis at adolescent/adult stages are unknown. Neuregulin 1 (NRG1) and its receptor ErbB4 represent schizophrenia-associated susceptibility factors that closely interact with NMDAR. To determine the neuronal populations implicated in “late” NMDAR-driven psychosis, we analyzed the effect of the inducible ablation of NMDARs in ErbB4-expressing cells in mice during late adolescence using a pharmacogenetic approach. Interestingly, the tamoxifen-inducible NMDAR deletion during this late developmental stage did not induce behavioral alterations resembling depression, schizophrenia or anxiety. Our data indicate that post-adolescent NMDAR deletion, even in a wider cell population than parvalbumin-positive interneurons, is also not sufficient to generate behavioral abnormalities resembling psychiatric disorders. Other neuronal substrates that have to be revealed by future studies, may underlie post-adolescent NMDAR-driven psychosis.

Sustained Biochemical Signaling and Contact Guidance by Electrospun Bicomponents as Promising Scaffolds for Nerve Tissue Regeneration.

Electrospun fibers are excellent delivery vehicles enabling a sustained release of growth factors to elicit favorable cell responses and are increasingly used in tissue engineering. Scaffolds with specific physical/topographical features can also guide cell migration and maturation. Therefore, growth factor-loaded electrospun scaffolds with a designed topography are promising for tissue regeneration. In this investigation, aligned-fiber scaffolds composed of poly(lactic-co-glycolic acid) nanofibers incorporating a glial cell line-derived growth factor and poly (d,l-lactic acid) nanofibers incorporating a nerve growth factor were produced by electrospinning. The scaffolds provided an aligned fibrous topography and a dual release of growth factors. The rat pheochromocytoma cell (PC12 cell) response to produced non-woven and aligned-fiber scaffolds with/without growth factors was studied. The dual release of growth factors and topographical cues provided by aligned-fiber bicomponent scaffolds induced significant neurite extension, neuronal differentiation, and neurite alignment in a synergistic manner. The scaffolds with predesigned biochemical/topographical cues demonstrated in this study might be promising for nerve tissue repair.

Abstract 10935: Identification of Small-Molecule ERBB4 Agonists to Treat Heart Failure

Introduction: Heart failure is a complex syndrome with a poor prognosis. The neuregulin-1 (NRG1)/ERBB4 axis has been shown to be cardioprotective and is a possible target for heart failure therapy. Clinical trials with NRG1 are ongoing, but require intravenous administration. Our aim is to develop a small-molecule ERBB4 agonist with cardioprotective properties. Methods: A high-throughput diversity screening (HTS) of 10,240 compounds was performed on a cellular ERBB4/ERBB4 dimerization assay. Ligand binding of hit compounds was tested in co-administration studies with NRG1 (0.1 μM) and competition binding assays with fluorescently labeled NRG1 (0.03 μM). Selectivity, post-receptor phosphorylation, and cell toxicity of hit compounds were determined using Luminex® RTK phosphoprotein, U2OS ERBB2/ERBB3 dimerization and adenylate kinase assays. Biological effects were studied in vitro in different doses (4-32 μM) on TGF-β-induced collagen synthesis by human dermal and atrial fibroblasts, and on angiotensin II (AngII, 1000 ng/kg/min)-induced left ventricular (LV) remodeling in mice with 2 selected hit compounds (83 μg/kg/h), administrated through osmotic pumps (N=4-5/group). After 28 days, left ventricular myocardial fibrosis and cardiomyocyte cross sectional area (CSA) were analyzed. Results: The HTS resulted in 8 similar pyrimidine derivatives inducing ERBB4/ERBB4 but not ERBB2/ERBB3 dimerization (Emax 9 to 33% relative to NRG1, and EC50 6x10 -6 M to 2x10 -7 M). Two compounds were excluded due to in vitro toxicity. The other 6 compounds were non-toxic and induced ERBB4, but not ERBB1, ERBB2 or ERBB3 phosphorylation in EFO-21 cells. Competition assays showed allosteric binding, and co-administration studies with NRG1 revealed that compounds potentiated NRG1-induced ERBB4 receptor dimerization up to 2.5 fold. Selected compounds showed significant dose-dependent antifibrotic effects in cultured human atrial and dermal fibroblasts and significantly attenuated AngII-induced LV myocardial fibrosis by 76%±26%, whereas cardiomyocyte CSA was not affected. Conclusions: We identified novel pyrimidine derivative small-molecule ERBB4 agonists with antifibrotic properties, both in vitro and in a mouse model of AngII-induced LV fibrosis.

Abstract PO-131: The role of liver endothelium on pancreatic cancer growth

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate among major cancers in the United States, and the 5-year survival rate for patients with metastatic PDAC (mPDAC) is only at 3%. Past studies have shown that the paracrine secretion of soluble factors by endothelial cells (ECs) created a unique niche and promoted the survival of cancer cells (cell growth or chemoresistance) in other types of cancer. The liver is the main site of distant metastases in mPDAC, but the influence of the liver EC microenvironment on mPDAC has not been elucidated. In this study, we determined the paracrine effects of liver ECs on the survival of PDAC and identify involved mechanism. Methods: Primary liver ECs were isolated from non-neoplastic liver tissues to mimic the liver EC microenvironment. Conditioned medium (CM) from liver ECs were collected and then applied to PDAC cells, with CM from PDAC as control CM. Effects of CM on PDAC cell proliferation were measured by the MTT assay. Changes in phosphorylation of receptor tyrosine kinases (RTK) between PDAC CM and EC CM treated PADC cells were determined by a Phospho-RTK Array kit and then validated by Western blotting. Involved RKTs were blocked by antibodies for determining their roles in mediating EC effects on PDAC cells. Lastly, A xenograft tumor model was used to establish PDAC tumors and then treated xenograft mice were subcutaneously injected by either PADC CM or EC CM, and tumors growth was monitored and recorded to evaluate the effect of ECs on PADCs. Results: Compared to PDAC CM, EC CM promoted proliferation in 4 different PDAC cells. We found that human epidermal growth factor receptor 3 (HER3 or ERBB3) was only expressed and activated in BxPC-3 cells (HER3+ve), in which the HER3-AKT signaling pathway was activated by EC CM. Furthermore, blocking HER3 activation with a humanized HER3 antibody, seribantumab, completely blocked EC CM-induced AKT activation and cell proliferation. Moreover, depletion of neuregulin (NRG) from EC CM attenuated HER3-AKT activation and indicated that EC-secreted NRG might play a role in promoting PDAC growth. It is interesting that ERK activation was also observed but was not affected by HER3 inhibition. It implied that EGFR signaling pathway might also be involved in EC CM induced PDAC cell growth. Moreover, the combination of cetuximab, trastuzumab and seribantumab yielded the best inhibitory ability on EC CM promoted cell growth as compared to antibody alone or the combination of two of them. Finally, EC CM promoted PDAC tumor growth was also observed in BxPC-3 derived xenograft mouse model. Conclusions: Our results demonstrated that liver EC-secreted factors promoted PDAC growth either in vitro or in vivo, and HER3 was expressed in a subset of PDAC cells and mediated EC-induced proliferation. Moreover, EGFR pathway may also play a role in EC induced cell growth and needs to be addressed in the future study. Our findings suggest a potential of using the combination of HER antibodies/inhibitors for treating patients with HER3+ve mPDACs. Citation Format: Wei Zhang, Michel’le Wright, Moeez Rathore, Ali Vaziri-Gohar, Jordan Winter, Rui Wang. The role of liver endothelium on pancreatic cancer growth [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-131.

Neuregulin-1 enhances cell-cycle activity, delays cardiac fibrosis, and improves cardiac performance in rat pups with right ventricular pressure load

ObjectivesRight ventricular (RV) failure is a leading cause of death in patients with congenital heart disease. RV failure is kept at bay during childhood. Limited proliferation of cardiomyocytes is present in the postnatal heart. We propose that cardiomyocyte proliferation improves RV adaptation to pressure load (PL). We studied adaptation in response to increased RV PL and the role of increased cardiomyocyte cell cycle activity (CCA) in rat pups growing into adulthood. MethodsWe induced RV PL at day of weaning in rats (3 weeks; 30-40 g) by pulmonary artery banding and followed rats into adulthood (300 g). We performed histological analyses and RNA sequencing analysis. To study the effects of increased cardiomyocyte cell cycle activity, we administered neuregulin-1 (NRG1), a growth factor involved in cardiac development. ResultsPL induced an increase in CCA, with subsequent decline of CCA (sham/PL at 4 weeks: 0.14%/0.83%; P = .04 and 8 weeks: 0.00%/0.00%; P = .484) and cardiac function (cardiac index: control/PL 4 weeks: 4.41/3.29; P = .468 and 8 weeks: 3.57/1.44; P = .024). RNA sequencing analysis revealed delayed maturation and increased CCA pathways. NRG1 stimulated CCA (PL vehicle/NRG1 at 2 weeks: 0.62%/2.28%; P = .003), improved cardiac function (cardiac index control vs vehicle/NRG1 at 2 weeks: 4.21 vs 3.07/4.17; P = .009/.705) and postponed fibrosis (control vs vehicle/NRG1 at 4 weeks: 1.66 vs 4.82%/2.97%; P = .009/.078) in RV PL rats during childhood. ConclusionsRV PL during growth induces a transient CCA increase. Further CCA stimulation improves cardiac function and delays fibrosis. This proof-of-concept study shows that stimulation of CCA can improve RV adaptation to PL in the postnatal developing heart and might provide a new approach to preserve RV function in patients with congenital heart disease.

Sustained ErbB Activation Causes Demyelination and Hypomyelination by Driving Necroptosis of Mature Oligodendrocytes and Apoptosis of Oligodendrocyte Precursor Cells.

Oligodendrocytes are vulnerable to genetic and environmental insults and its injury leads to demyelinating diseases. The roles of ErbB receptors in maintaining the CNS myelin integrity are largely unknown. Here, we overactivate ErbB receptors that mediate signaling of either neuregulin (NRG) or epidermal growth factor (EGF) family growth factors and found their synergistic activation caused deleterious outcomes in white matter. Sustained ErbB activation induced by the tetracycline-dependent mouse tool Plp-tTA resulted in demyelination, axonal degeneration, oligodendrocyte precursor cell (OPC) proliferation, astrogliosis, and microgliosis in white matter. Moreover, there was hypermyelination before these inflammatory pathologic events. In contrast, sustained ErbB activation induced by another tetracycline-dependent mouse tool Sox10+/rtTA caused hypomyelination in the corpus callosum and optic nerve, which appeared to be a developmental deficit and did not associate with OPC regeneration, astrogliosis, or microgliosis. By tracing the differentiation states of cells expressing tetracycline-controlled transcriptional activator (tTA)/reverse tTA (rtTA)-dependent transgene or pulse-labeled reporter proteins in vitro and in vivo, we found that Plp-tTA targeted mainly mature oligodendrocytes (MOs), whereas Sox10+/rtTA targeted OPCs and newly-formed oligodendrocytes (NFOs). The distinct phenotypes of mice with ErbB overactivation induced by Plp-tTA and Sox10+/rtTA consolidated their nonoverlapping targeting preferences in the oligodendrocyte lineage, and enabled us to demonstrate that ErbB overactivation in MOs induced necroptosis that caused inflammatory demyelination, whereas in OPCs induced apoptosis that caused noninflammatory hypomyelination. Early interference with aberrant ErbB activation ceased oligodendrocyte deaths and restored myelin development in both mice. This study suggests that aberrant ErbB activation is an upstream pathogenetic mechanism of demyelinating diseases, providing a potential therapeutic target.SIGNIFICANCE STATEMENT Primary oligodendropathy is one of the etiologic mechanisms for multiple sclerosis, and oligodendrocyte necroptosis is a pathologic hallmark in the disease. Moreover, the demyelinating disease is now a broad concept that embraces schizophrenia, in which white matter lesions are an emerging feature. ErbB overactivation has been implicated in schizophrenia by genetic analysis and postmortem studies. This study suggests the etiologic implications of ErbB overactivation in myelin pathogenesis and elucidates the pathogenetic mechanisms.

Association of Neuregulin Levels and Neuregulin-1 Polymorphism with Short-term Morbidities in Preterm Neonates

Background Premature birth is linked to neonatal morbidity and mortality worldwide. Neuregulin (NRG) is a trophic factor from the growth factor (GF) of a transmembrane polypeptide, encoded by four different genes, including NRG-1 which acts as an endogenous protector in fetal development. Decreased levels of NRG-1 affect several organs. The relationship between NRG-1 polymorphism and the outcome of neonatal development has been widely studied. There are no studies that have assessed NRG-1 levels and NRG-1 rs35753505 C/T polymorphism in preterm neonates, as well as its association with short-term morbidities in Indonesia.Methods This cross-sectional study was conducted on preterm neonates with the gestational age of 32-36 weeks in Medan, North Sumatera, Indonesia, from December 2017 to December 2018. It aimed to evaluate the association of NRG-1 levels and NRG1 polymorphism with short-term morbidities. Samples were obtained from cord blood specimens. Enzyme-linked immunosorbent assay (ELISA) was used to determine NRG-1 levels, and NRG-1 polymorphism was sequenced by polymerase chain reaction (PCR). Observations in preterm neonates were made during the first 72 h to assess short-term morbidities.Results During the study period, 48 cord blood specimens from preterm neonates were found eligible for analysis. Preterm neonates with low NRG-1 levels had a 10-times higher risk of developing short-term morbidities. The presence of CC and CT genotypes increased the risk of developing short-term morbidities 13.33 times (P=0.003) and 6.19 times (P=0.019), respectively. The presence of the C allele in subjects' genotype increased the risk of short-term morbidities 4.04 times (P=0.001), compared to those with T allele.Conclusion As evidenced by the obtained results, preterm neonates with low NRG-1 levels had a higher risk of developing short-term morbidities. Furthermore, there was a significant association between NRG-1 rs35753505 C/T polymorphism and short-term morbidities.

Discovering of Genomic Variations Associated to Growth Traits by GWAS in Braunvieh Cattle.

A genome-wide association study (GWAS) was performed to elucidate genetic architecture of growth traits in Braunvieh cattle. Methods: The study included 300 genotyped animals by the GeneSeek® Genomic Profiler Bovine LDv.4 panel; after quality control, 22,734 SNP and 276 animals were maintained in the analysis. The examined phenotypic data considered birth (BW), weaning (WW), and yearling weights. The association analysis was performed using the principal components method via the egscore function of the GenABEL version 1.8-0 package in the R environment. The marker rs133262280 located in BTA 22 was associated with BW, and two SNPs were associated with WW, rs43668789 (BTA 11) and rs136155567 (BTA 27). New QTL associated with these liveweight traits and four positional and functional candidate genes potentially involved in variations of the analyzed traits were identified. The most important genes in these genomic regions were MCM2 (minichromosome maintenance complex component 2), TPRA1 (transmembrane protein adipocyte associated 1), GALM (galactose mutarotase), and NRG1 (neuregulin 1), related to embryonic cleavage, bone and tissue growth, cell adhesion, and organic development. This study is the first to present a GWAS conducted in Braunvieh cattle in Mexico providing evidence for genetic architecture of assessed growth traits. Further specific analysis of found associated genes and regions will clarify its contribution to the genetic basis of growth-related traits.

Neuregulin 1 Gene (NRG1). A Potentially New Targetable Alteration for the Treatment of Lung Cancer.

Simple SummaryTreatment in oncology has and will keep evolving into an agnostic approach where therapies are guided more towards the identification and targeting of genetic abnormalities and less by organ of origin of the cancer, as has been done for decades. With every genetic abnormality being identified as a target, the pharmaceutical development of medications targeting these genes has grown, leading to better survival rates, quality of life and a bigger interest in finding new targets. Lung cancer is one of the best examples where targetable genetic abnormalities have led to substantial survival differences compared to patients undergoing empirical conventional chemotherapy. Translocations in the neuregulin 1 gene (NRG1) are one of many gene fusions that are becoming clinically significant, and it has the potential to become a targetable gene with ongoing clinical trials already in Europe and the US. This review aims to portray the importance and latest developments regarding this new fusion in lung cancer treatment.Oncogenic gene fusions are hybrid genes that result from structural DNA rearrangements, leading to unregulated cell proliferation by different mechanisms in a wide variety of cancer. This has led to the development of directed therapies to antagonize a variety of mechanisms that lead to cell growth or proliferation. Multiple oncogene fusions are currently targeted in lung cancer treatment, such as those involving ALK, RET, NTRK and ROS1 among many others. Neuregulin (NRG) gene fusion has been described in the development of normal tissue as well as in a variety of diseases, such as schizophrenia, Hirschsprung’s disease, atrial fibrillation and, most recently, the development of various types of solid tumors, such as renal, gastric, pancreatic, breast, colorectal and, more recently, lung cancer. The mechanism for this is that the NRG1 chimeric ligand leads to aberrant activation of ERBB2 signaling via PI3K-AKT and MAPK cellular cascades, leading to cell division and proliferation. Details regarding the incidence of these gene rearrangements are lacking. Limited case reports and case series have evaluated their clinicopathologic features and prognostic significance in the lung cancer population. Taking this into account, NRG1 could become a targetable alteration in selected patients. This review highlights how the knowledge of new molecular mechanisms of NRG1 fusion may help in gaining new insights into the molecular status of lung cancer patients and unveil a novel targetable molecular marker.