Receptor Tyrosine Kinases as Candidate Prognostic Biomarkers and Therapeutic Targets in Meningioma.

Meningioma (MGM) is the most common type of intracranial tumor in adults. The validation of novel prognostic biomarkers to better inform tumor stratification and clinical prognosis is urgently needed. Many molecular and cellular alterations have been described in MGM tumors over the past few years, providing a rational basis for the identification of biomarkers and therapeutic targets. The role of receptor tyrosine kinases (RTKs) as oncogenes, including those of the ErbB family of receptors, has been well established in several cancer types. Here, we review histological, molecular, and clinical evidence suggesting that RTKs, including the epidermal growth factor receptor (EGFR, ErbB1), as well as other members of the ErbB family, may be useful as biomarkers and therapeutic targets in MGM.

Genomic and transcriptomic analysis of pituitary adenomas reveals the impacts of copy number variations on gene expression and clinical prognosis among prolactin-secreting subtype.

Pituitary adenomas (PAs) are slow growing and benign primary intracranial tumors that often cause occupying effects or endocrine symptoms. PAs can be classified into various subtypes according to hormone secretion. Although widespread transcriptional alterations that cause aberrant hormone secretion have been characterized, the impact of genomic variations on transcriptional alterations is unclear due to the rare occurrence of single-nucleotide variations in PA. In this study, we performed whole-genome sequencing (WGS) on 76 PA samples across three clinical subtypes (PRL-PAs; GH-PAs, and NFPAs); transcriptome sequencing (RNA-seq) of 54 samples across these subtypes was also conducted. Nine normal pituitary tissues were used as controls. Common and subtype-specific transcriptional alterations in PAs were identified. Strikingly, widespread genomic copy number amplifications were discovered for PRL-PAs, which are causally involved in transcriptomic changes in this subtype. Moreover, we found that the high copy number variations (CNVs) in PRL-PA cause increased prolactin production, drug resistance and proliferative capacity, potentially through key genes with copy number amplification and transcriptional activation, such as BCAT1. This study provides insight into how genomic CNVs affect the transcriptome and clinical outcomes of PRL-PA and sheds light on the development of potential therapeutics for aberrantly activated targets.

Clozapine-dependent inhibition of EGF/neuregulin receptor (ErbB) kinases.

Clozapine is an antipsychotic agent prescribed to psychotic patients exhibiting tolerance and/or resistance to the conventional antipsychotic medications that mainly drive monoamine antagonism. As the pharmacological fundamentals of its unique antipsychotic profile have been unrevealed, here, we attempted to obtain hints at this question. Here, we found that clozapine directly acts on ErbB kinases to downregulate epidermal growth factor (EGF)/neuregulin signaling. In cultured cell lines and cortical neurons, EGF-triggered ErbB1 phosphorylation was diminished by 30 µM clozapine, but not haloperidol, risperidone, or olanzapine. The neuregulin-1-triggered ErbB4 phosphorylation was attenuated by 10 µM clozapine and 30 µM haloperidol. We assumed that clozapine may directly interact with the ErbB tyrosine kinases and affect their enzyme activity. To test this assumption, we performed in vitro kinase assays using recombinant truncated ErbB kinases. Clozapine (3-30 µM) significantly decreased the enzyme activity of the truncated ErbB1, B2, and B4 kinases. Acute in vivo administration of clozapine (20 mg/kg) to adult rats significantly suppressed the basal phosphorylation levels of ErbB4 in the brain, although we failed to detect effects on basal ErbB1 phosphorylation. Altogether with the previous findings that quinazoline inhibitors for ErbB kinases harbor antipsychotic potential in animal models for schizophrenia, our present observations suggest the possibility that the micromolar concentrations of clozapine can attenuate the activity of ErbB receptor kinases, which might illustrate a part of its unique antipsychotic psychopharmacology.

A fungicide miconazole ameliorates tri-o-cresyl phosphate-induced demyelination through inhibition of ErbB/Akt pathway.

Organophosphorus compound (OP)-induced delayed neuropathy (OPIDN) is characterized by distal axonal degeneration and demyelination of the central and peripheral axons, which leads to progressive muscle weakness, ataxia and paralysis in several days after OP intoxication. This study aimed to investigate the possible use of an imidazole fungicide miconazole as a novel therapy for OPIDN. Adult hens, the most commonly used animal models in OPIDN studies, were orally given tri-o-cresyl phosphate (TOCP). We showed that miconazole, which was administered daily to hens beginning on the 7th day after TOCP exposure, drastically ameliorated the neurotoxic symptoms and histopathological damages in spinal cord and sciatic nerves. Mechanistically, miconazole inhibited the TOCP-induced activation of ErbB/Akt signaling, and enhanced the myelin basic protein (MBP) expression. In a glial cell model sNF96.2 cells, miconazole restored the TOCP-inhibited MBP expression, and promoted cell differentiation as well as cell migration by inhibiting the activation of ErbB/Akt signaling pathway. In sum, miconazole, a synthetic imidazole fungicide, could ameliorate the symptoms and histopathological changes of OPIDN, probably by promoting glial cell differentiation and migration to enhance myelination via inhibiting the activation of ErbB/Akt. Thus, miconazole is a promising candidate therapy for the clinical treatment of OPIDN.

Astraglaus polysaccharide protects diabetic cardiomyopathy by activating NRG1/ErbB pathway.

Diabetic cardiomyopathy (DCM) is one of the main cardiac complications among diabetic patients. According to previous studies, the pathogenesis of DCM is associated with oxidative stress, apoptosis and proliferation of local cardiac cells. It showed, NRG1 can improve the function of mitochondria, and thereby, increasing proliferation and decreasing apoptosis of cardiac muscle cell via ErbB/AKT signaling, also, exert antioxidative function. Besides, NRG1/ErbB pathway was impaired in the DCM model which suggested this signaling played key role in DCM. Astraglaus polysaccharide (APS), one of the active components of Astragalus mongholicus, showed striking antioxidative effect. Here, in this study, our data showed that APS can promote proliferation and decrease apoptosis in AGE-induced DCM cell model, besides, APS can decrease intracellular ROS level, increase activity of SOD, GSH-Px and lower level of MDA and NO in DCM cell model, indicating APS exerted antioxidative function in DCM model cells. Besides, western blot results revealed APS induced NRG1 expressing and the phosphorylation level of ErbB2/4. In addition, the elevated NRG1 promoted AKT and PI3k phosphorylation which indicated APS may exert its function by NRG1/ErbB and the downstream AKT/PI3K signaling. Canertinib is ErbB inhibitor. The effect of APS on proliferation, apoptosis, antioxidation and NRG1/ErbB pathway was partly abolished after the cells were co-treated with APS and canertinib. Taken together, these results suggested APS may display its protective function in DCM cells by activating NGR1/ErbB signaling pathway. And our study increased potential for prevention and therapy to DCM.

Differential recruitment of CD44 isoforms by ErbB ligands reveals an involvement of CD44 in breast cancer.

Members of the CD44 family of transmembrane glycoproteins control cell signaling pathways from numerous cell surface receptors, including receptor tyrosine kinases (RTKs). The decisive factor (ligand, RTKs or both) that controls the recruitment of specific CD44 isoforms is still unknown. We investigated this question by using the EGFR signaling pathway, in which one receptor can be activated by a broad range of ligands. By means of siRNA-mediated downregulation of CD44 expression and blocking experiments, we identified CD44v6 as a co-receptor for EGF- and ER-induced ErbB1 activation and for NRG1-induced ErbB3 and ErbB4 activation. In contrast, TGFα is independent of all CD44 isoforms, even though it addresses the same receptor pairs as EGF. Moreover, the heparin-sulfated CD44v3 isoform is required for HB-EGF-induced EGFR signaling. These data suggest that specific CD44 isoforms are recruited in a ligand-dependent manner as co-receptors in the EGFR signaling pathways and that the specificity is determined by the ligand and not by the receptors themselves. The in vivo relevance of this interplay between CD44 isoforms and EGFR ligands is underlined by the decreased metastatic spreading of mammary carcinomas in mice treated with a CD44v6-specific peptide. Most importantly, we found a clear correlation between the presence of CD44v6/ErbB1 complexes in breast cancer patients and lymph node metastases.

miRNAs: mediators of ErbB family targeted therapy resistance.

The ErbB/HER tyrosine kinase receptors family plays a key regulatory role in different cellular processes by activating several signaling pathways. In different tumor types, mutations or overexpression of the ErbB family members are a common feature, which led to the development of targeted therapies against this receptors. Although with this kind of treatment we are heading to a more personalized medicine, the development of acquired resistance is still an issue, therefore, several studies focused on discovering the mechanisms behind it. More recently, miRNAs have been described as important mediators of acquired resistance, specifically, acquired resistance to ErbB family targeted therapies. Ultimately, miRNA-based therapeutics using exosomes as a drug delivery model can revolutionize today's approach of cancer treatment.

The hibernating South American marsupial, Dromiciops gliroides, displays torpor-sensitive microRNA expression patterns.

When faced with adverse environmental conditions, the marsupial Dromiciops gliroides uses either daily or seasonal torpor to support survival and is the only known hibernating mammal in South America. As the sole living representative of the ancient Order Microbiotheria, this species can provide crucial information about the evolutionary origins and biochemical mechanisms of hibernation. Hibernation is a complex energy-saving strategy that involves changes in gene expression that are elicited in part by microRNAs. To better elucidate the role of microRNAs in orchestrating hypometabolism, a modified stem-loop technique and quantitative PCR were used to characterize the relative expression levels of 85 microRNAs in liver and skeletal muscle of control and torpid D. gliroides. Thirty-nine microRNAs were differentially regulated during torpor; of these, 35 were downregulated in liver and 11 were differentially expressed in skeletal muscle. Bioinformatic analysis predicted that the downregulated liver microRNAs were associated with activation of MAPK, PI3K-Akt and mTOR pathways, suggesting their importance in facilitating marsupial torpor. In skeletal muscle, hibernation-responsive microRNAs were predicted to regulate focal adhesion, ErbB, and mTOR pathways, indicating a promotion of muscle maintenance mechanisms. These tissue-specific responses suggest that microRNAs regulate key molecular pathways that facilitate hibernation, thermoregulation, and prevention of muscle disuse atrophy.

Dysregulation of Neuregulin-1/ErbB signaling in the prefrontal cortex and hippocampus of rats exposed to chronic unpredictable mild stress.

Exposure to chronic stress increases the likelihood of developing depression, but the underlying mechanisms remain equivocal. While recent evidence has indicated that Neuregulin-1 (NRG1) and its ErbB receptors play an essential role in neural development and function, and NRG1 has emerged as a novel modulator involved in the response of brain to stress, there is limited evidence concerning the effects of chronic stress exposure on NRG1/ErbB signaling. To fill this critical gap, we examined the protein expression of NRG1 and ErbB receptors in the brain of rats following chronic unpredictable mild stress (CUMS) exposure. After 6weeks of CUMS procedures, the rats were induced to a depression-like state. The stressed rats displayed elevated expression of NRG1 and phosphorylated ErbB4 (pErbB4) in the prefrontal cortex, whereas ErbB2 and pErbB2 were inhibited. In the hippocampus, CUMS also attenuated activation of the both ErbB receptors and suppressed the downstream Akt and ERK phosphorylation. Meanwhile, administration of sertraline enhanced NRG1/ErbB signaling and partly normalized the stress-induced behavioral changes and the disturbances of NRG1/ErbB system in CUMS rats. Combined, our data firstly showed the aberrant changes of NRG1/ErbB system in the brain of the animal model of depression, providing new evidence for the involvement of NRG1/ErbB pathway in the development and treatment of depression.

Neuregulin1 and ErbB expression in the uninjured and regenerating olfactory mucosa.

Neuregulin1, a protein involved in signaling through the ErbB receptors, is required for the proper development of multiple organ systems. A complete understanding of the expression profile of Neuregulin1 is complicated by the presence of multiple isoform variants that result from extensive alternative splicing. Remarkably, these numerous protein products display a wide range of divergent functional roles, making the characterization of tissue-specific isoforms critical to understanding signaling. Recent evidence suggests an important role for Neuregulin1 signaling during olfactory epithelium development and regeneration. In order to understand the physiological consequences of this signaling, we sought to identify the isoform-specific and cell type-specific expression pattern of Neuregulin1 in the adult olfactory mucosa using a combination of RT-qPCR, FACS, and immunohistochemistry. To complement this information, we also analyzed the cell-type specific expression patterns of the ErbB receptors using immunohistochemistry. We found that multiple Neuregulin1 isoforms, containing predominantly the Type I and Type III N-termini, are expressed in the uninjured olfactory mucosa. Specifically, we found that Type III Neuregulin1 is highly expressed in mature olfactory sensory neurons and Type I Neuregulin1 is highly expressed in duct gland cells. Surprisingly, the divergent localization of these Neuregulin isoforms and their corresponding ErbB receptors does not support a role for active signaling during normal turnover and maintenance of the olfactory mucosa. Conversely, we found that injury to the olfactory epithelium specifically upregulates the Neuregulin1 Type I isoform bringing the expression pattern adjacent to cells expressing both ErbB2 and ErbB3 which is compatible with active signaling, supporting a functional role for Neuregulin1 specifically during regeneration.

The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

The metalloproteinase ADAM17 activates ErbB signalling by releasing ligands from the cell surface, a key step underlying epithelial development, growth and tumour progression. However, mechanisms acutely controlling ADAM17 cell-surface availability to modulate the extent of ErbB ligand release are poorly understood. Here, through a functional genome-wide siRNA screen, we identify the sorting protein PACS-2 as a regulator of ADAM17 trafficking and ErbB signalling. PACS-2 loss reduces ADAM17 cell-surface levels and ADAM17-dependent ErbB ligand shedding, without apparent effects on related proteases. PACS-2 co-localizes with ADAM17 on early endosomes and PACS-2 knockdown decreases the recycling and stability of internalized ADAM17. Hence, PACS-2 sustains ADAM17 cell-surface activity by diverting ADAM17 away from degradative pathways. Interestingly, Pacs2-deficient mice display significantly reduced levels of phosphorylated EGFR and intestinal proliferation. We suggest that this mechanism controlling ADAM17 cell-surface availability and EGFR signalling may play a role in intestinal homeostasis, with potential implications for cancer biology.

Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development, Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis.

Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight genes--fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a--had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a)-ErbB2/3-AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a-ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity and cell shape changes serves as a crucial mechanism of epithelial morphogenesis.

Axon-Schwann cell interactions during peripheral nerve regeneration in zebrafish larvae.

BACKGROUND: Peripheral nerve injuries can severely affect the way that animals perceive signals from the surrounding environment. While damage to peripheral axons generally has a better outcome than injuries to central nervous system axons, it is currently unknown how neurons re-establish their target innervations to recover function after injury, and how accessory cells contribute to this task. Here we use a simple technique to create reproducible and localized injury in the posterior lateral line (pLL) nerve of zebrafish and follow the fate of both neurons and Schwann cells. RESULTS: Using pLL single axon labeling by transient transgene expression, as well as transplantation of glial precursor cells in zebrafish larvae, we individualize different components in this system and characterize their cellular behaviors during the regenerative process. Neurectomy is followed by loss of Schwann cell differentiation markers that is reverted after nerve regrowth. We show that reinnervation of lateral line hair cells in neuromasts during pLL nerve regeneration is a highly dynamic process with promiscuous yet non-random target recognition. Furthermore, Schwann cells are required for directional extension and fasciculation of the regenerating nerve. We provide evidence that these cells and regrowing axons are mutually dependant during early stages of nerve regeneration in the pLL. The role of ErbB signaling in this context is also explored. CONCLUSION: The accessibility of the pLL nerve and the availability of transgenic lines that label this structure and their synaptic targets provides an outstanding in vivo model to study the different events associated with axonal extension, target reinnervation, and the complex cellular interactions between glial cells and injured axons during nerve regeneration.

Structure-function relationships of ErbB RTKs in the plasma membrane of living cells.

We review the states of the ErbB family of receptor tyrosine kinases (RTKs), primarily the EGF receptor (EGFR, ErbB1, HER1) and the orphan receptor ErbB2 as they exist in living mammalian cells, focusing on four main aspects: (1) aggregation state and distribution in the plasma membrane; (2) conformational features of the receptors situated in the plasma membrane, compared to the crystallographic structures of the isolated extracellular domains; (3) coupling of receptor disposition on filopodia with the transduction of signaling ligand gradients; and (4) ligand-independent receptor activation by application of a magnetic field.

miR-125a regulates cell cycle, proliferation, and apoptosis by targeting the ErbB pathway in acute myeloid leukemia.

microRNA profiling of acute myeloid leukemia patient samples identified miR-125a as being decreased. Current literature has investigated miR-125a's role in normal hematopoiesis but not within acute myeloid leukemia. Analysis of the upstream region of miR-125a identified several CpG islands. Both precursor and mature miR-125a increased in response to a de-methylating agent, Decitabine. Profiling revealed the ErbB pathway as significantly decreased with ectopic miR-125a. Either ectopic expression of miR-125a or inhibition of ErbB via Mubritinib resulted in inhibition of cell cycle proliferation and progression with enhanced apoptosis revealing ErbB inhibitors as potential novel therapeutic agents for treating miR-125a-low AML.

Central neuregulin-1/ErbB signaling modulates cardiac function via sympathetic activity in pressure overload-induced heart failure.

OBJECTIVES: Neuregulin-1 (NRG-1)/ErbB signaling in the heart is reported to have a crucial role in heart failure. We recently demonstrated that NRG-1 signaling has sympathoinhibitory effects in the brain cardiovascular control center. How this central signaling impacts sympathoexcitation in heart failure, however, is unknown. Here we examined the role of central NRG-1/ErbB signaling in modulating the sympathetic nervous system in pressure overload-induced heart failure. METHODS AND RESULTS: Pressure overload-induced heart failure was induced in Wistar-Kyoto rats by banding the abdominal aorta. Rats were followed up for 15 weeks. Compared to sham-operated rats, aortic-banded rats showed left ventricle (LV) hypertrophy, LV dilation, and LV dysfunction [reducing fractional shortening (%fractional shortening), increased LV end-diastolic pressure, decreased positive and negative pressure differential (±dp/dt(max))], and increased urinary norepinephrine excretion. Aortic banding led to reduced expression of NRG-1 in the brainstem at 10 weeks after banding and reduced expression of ErbB2 at 5 weeks, but did not affect ErbB4. Central administration of recombinant NRG-1ß at 5 weeks for 2 weeks attenuated LV hypertrophy, improved LV dilatation, prevented LV dysfunction (improvement of %fractional shortening and ±dp/dt(max), and reduction of LV end-diastolic pressure), and lowered urinary norepinephrine excretion at 10 weeks, and these effects were still observed at 15 weeks. CONCLUSION: NRG-1/ErbB signaling in the brainstem is impaired during the progression of pressure overload-induced heart failure. Activation of central NRG-1 signaling improves cardiac function through sympathoinhibition. These findings provide a new treatment concept and support the benefit of NRG-1 treatment in heart failure.

Preclinical and clinical development of afatinib: a focus on breast cancer and squamous cell carcinoma of the head and neck.

Aberrant signaling of the ErbB family of receptors plays an integral role in the tumorigenesis of many cancer types, including head and neck squamous cell carcinoma (HNSCC) and breast cancer (BC). Significant research efforts have focused on developing new treatments that target ErbB family members, with the last decade seeing the approval of small-molecule tyrosine kinase inhibitors and monoclonal antibodies that inhibit ErbB signaling. However, treatment resistance is an ever-growing problem and, therefore, new therapies are being investigated to overcome this hurdle. Afatinib is an irreversible ErbB family blocker that has demonstrated potent anti-tumor activity in preclinical models and has displayed clinical efficacy in patients with non-small-cell lung cancer, and activity in HNSCC and BC. Here, the preclinical and clinical development of afatinib in the treatment of non-small-cell lung cancer, HNSCC and BC is described in the context of currently approved agents.

Smooth muscle cells relay acute pulmonary inflammation via distinct ADAM17/ErbB axes.

In acute pulmonary inflammation, danger is first recognized by epithelial cells lining the alveolar lumen and relayed to vascular responses, including leukocyte recruitment and increased endothelial permeability. We supposed that this inflammatory relay critically depends on the immunological function of lung interstitial cells such as smooth muscle cells (SMC). Mice with smooth muscle protein-22α promotor-driven deficiency of the disintegrin and metalloproteinase (ADAM) 17 (SM22-Adam17(-/-)) were investigated in models of acute pulmonary inflammation (LPS, cytokine, and acid instillation). Underlying signaling mechanisms were identified in cultured tracheal SMC and verified by in vivo reconstitution experiments. SM22-Adam17(-/-) mice showed considerably decreased cytokine production and vascular responses in LPS- or acid-induced pulmonary inflammation. In vitro, ADAM17 deficiency abrogated cytokine release of primary SMC stimulated with LPS or supernatant of acid-exposed epithelial cells. This was explained by a loss of ADAM17-mediated growth factor shedding. LPS responses required ErbB1/epidermal growth factor receptor transactivation by TGFα, whereas acid responses required ErbB4 transactivation by neuregulins. Finally, LPS-induced pulmonary inflammation in SM22-Adam17(-/-) mice was restored by exogenous TGFα application, confirming the involvement of transactivation pathways in vivo. This highlights a new decisive immunological role of lung interstitial cells such as SMC in promoting acute pulmonary inflammation by ADAM17-dependent transactivation.