Hippocampal function is important for learning and memory, and dysfunction of the hippocampus has been linked to the pathophysiology of neuropsychiatric diseases such as schizophrenia. Neuregulin1 (NRG1) and ErbB4, two susceptibility genes for schizophrenia, reportedly modulate long-term potentiation (LTP) at hippocampal Schaffer collateral (SC)-CA1 synapses. However, little is known regarding the contribution of hippocampal NRG1/ErbB4 signaling to learning and memory function. Here, quantitative real-time PCR and Western blotting were used to assess the mRNA and protein levels of NRG1 and ErbB4. Pharmacological and genetic approaches were used to manipulate NRG1/ErbB4 signaling, following which learning and memory behaviors were evaluated using the Morris water maze, Y-maze test, and the novel object recognition test. Spatial learning was found to reduce hippocampal NRG1 and ErbB4 expression. The blockade of NRG1/ErbB4 signaling in hippocampal CA1, either by neutralizing endogenous NRG1 or inhibiting/ablating ErbB4 receptor activity, enhanced hippocampus-dependent spatial learning, spatial working memory, and novel object recognition memory. Accordingly, administration of exogenous NRG1 impaired those functions. More importantly, the specific ablation of ErbB4 in parvalbumin interneurons also improved learning and memory performance. The manipulation of NRG1/ErbB4 signaling in the present study revealed that NRG1/ErbB4 activity in the hippocampus is critical for learning and memory. These findings might provide novel insights on the pathophysiological mechanisms of schizophrenia and a new target for the treatment of Alzheimer's disease, which is characterized by a progressive decline in cognitive function.

BACKGROUND

The Cancer Genome Atlas analysis revealed that somatic EGFR, receptor tyrosine-protein kinase erbB-2 (ERBB2), Erb-B2 receptor tyrosine kinase 3 (ERBB3) and Erb-B2 receptor tyrosine kinase 4 (ERBB4) gene mutations (ERBB family mutations) occur alone or co-occur with somatic mutations in the gene encoding the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (PIK3CA) in 19% of human epidermal growth factor receptor 2 (HER2)-positive breast cancers. Because ERBB family mutations can activate the PI3K/AKT pathway and likely have similar canonical signalling effects to PI3K pathway mutations, we investigated their combined impact on response to neoadjuvant HER2-targeted therapies.

METHODS

Baseline tumour biopsies were available from 74 patients with HER2-positive breast cancer who were enrolled in the phase II TCHL neoadjuvant study (ICORG 10-05) assessing TCH (docetaxel, carboplatin, trastuzumab) (n = 38) versus TCL (docetaxel, carboplatin, lapatinib) (n = 10) versus TCHL (docetaxel, carboplatin, trastuzumab, lapatinib) (n = 40), each for six cycles. Activating mutations in PIK3CA and ERBB family genes were identified using mass spectrometry-based genotyping. Phosphatase and tensin homolog (PTEN) expression was assessed by immunohistochemistry.

RESULTS

PIK3CA and/or ERBB family mutations were detected in 23 (31.1%) tumour samples tested, whereas PTEN expression was low in 31.1% of cases tested. Mutation frequency was similar in each treatment arm (31.3% in TCH arm, 30% in TCL arm and 31.3% in TCHL arm) and was not influenced by oestrogen receptor (ER) status (27.6% in ER-negative patients, 33.3% in ER-positive patients) or progesterone receptor (PR) status (32.6% in PR-negative patients, 29% in PR-positive patients). There was no significant difference in pathological complete response (pCR) rates between 47 patients with wild-type (WT) tumours and 22 patients whose tumours carried mutations (in either PIK3CA or ERBB family genes) (42.5% vs. 54.5%; p = 0.439). Similarly, there was no significant difference in pCR rates between patients with PIK3CA/ERBB family mutated/PTEN-low (i.e., PI3K-activated) tumours and patients without PI3K activation (50% vs. 44%; p = 0.769). However, in the TCHL (but not the TCH) group, the pCR rate was higher for 9 patients with PIK3CA/ERBB family mutated tumours than for 20 patients with PIK3CA/ERBB family WT tumours (77.8% vs. 35%; p = 0.05).

CONCLUSIONS

Our results indicate that patients who receive neoadjuvant TCHL and have PIK3CA/ERBB family mutated tumours may be more likely to have a pCR than patients with WT tumours.

BACKGROUND

ClinicalTrials.gov, NCT01485926 . Registered on 2 December 2011.

The ErbB signalling network plays a crucial role in the growth and progression of several cancers, including colorectal cancer (CRC), and includes potentially drug-targetable genes. Oncogenic activation of the ErbB pathway by mutations and focal amplifications have emerged recently as an important predictive marker of the prognosis of CRC patients. However, in contrast to genetic events, little is known about epigenetic alternations of ErbB-associated genes and their impact on gene expression. Genome-wide methylation in sporadic CRCs (n = 12) paired with adjacent normal tissues have been previously analysed by Illumina Infinium HumanMethylation27 (HM27) at 27,578 CpG sites. For confirmation of our initial genome-wide analysis, we used a published HM27 dataset (GSE25062). Subsequently, CpG island methylation of selected ErbB pathway-associated genes was assessed on 233 CRC samples using methylation-sensitive polymerase chain reaction (MS-PCR) and analysed along with various genetic factors associated with CRC [epigenotype, BRAF and KRAS mutations, microsatellite instability (MSI)]. Methylation and expression integration was performed using published datasets including 25 pairs of CRC and normal colon tissues (GSE25062 and GSE25070), and confirmed with real-time PCR. Our previous microarray-based genome-wide DNA methylation analysis of 12 CRCs revealed that four ErbB-associated genes (PIK3CD, PKCΒ, ERBB4, ) were differentially methylated in CRCs. This was further confirmed by statistical re-analysis of an HM27 dataset (GSE25062). Frequent methylation at these loci in tumours was subsequently confirmed by MS-PCR (63%, 43%, 43% and 92%, respectively). Hypermethylation of PKCΒ associated with KRAS mutation (p = 0.04), whereas hypermethylation of ERBB4 associated with high-methylation epigenotypes (HME), BRAF mutation and MSI (p = 0.001, 0.002 and 0.0002, respectively). One of the four analysed genes (PKCΒ) was significantly downregulated in CRC tissue, as revealed by real-time PCR and re-analysis of the GSE25062 and GSE25070 datasets. After careful re-analysis of published methylation and expression data, we conclude that methylation of ERBB4, PAK7 and PIK3CD has no functional role in CRC carcinogenesis. In contrast, methylation seems to have a potential impact on the biology of colorectal tumours by negatively modulating the expression of PKCΒ. Importantly, the relationship between DNA methylation of PKCΒ and gene expression may warrant further attention in the context of colon cancer chemoprevention and anti-cancer therapy.

Neuregulin-1 (NRG1) is a trophic and differentiation factor that signals through ErbB receptor tyrosine kinases to regulate nervous system development. Previous studies have demonstrated that NRG1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons of the hippocampus and frontal cortex; however, immunohistochemical and genetic analyses strongly suggest these effects are indirect and mediated via ErbB4 receptors on GABAergic interneurons. Here, we used cultured cerebellar granule cells (CGCs) that express ErbB4 to analyze the cell-autonomous effects of NRG1 stimulation on glutamatergic function. These cultures have the advantage that they are relatively homogenous and consist primarily of granule neurons that express ErbB4. We show that acute NRG1 treatment does not affect whole-cell AMPA or NMDA receptor (NMDAR) mediated currents in CGCs at 10-12 days in vitro. NRG1 also does not affect the frequency or amplitude of spontaneous AMPAR or NMDAR mediated miniature excitatory post-synaptic currents (mEPSCs). To further investigate the effects of NRG1 on activity-dependent plasticity of glutamatergic synapses in CGCs, we characterized the effects of high-glyine/0 Mg(2+) (which activates synaptic NMDARs) on AMPAR-mEPSC frequency and amplitude. We show that high-glycine induces a form of chemical long-term potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency but not amplitude. Moreover, NRG1 induces a decrease in AMPAR-mEPSC frequency following chemLTP, but does not affect AMPAR-mEPSC amplitude. CGCs in our cultures conditions express low levels of GluR1, in contrast to dissociated hippocampal cultures, but do express the long isoform of GluR4. This study provides first evidence that (1) high-glycine can induce plasticity at glutamatergic synapses in CGCs, and (2) that acute NRG1/ErbB-signaling can regulate glutamatergic plasticity in CGCs. Taken together with previous reports, our results suggest that, similar to Schaeffer collateral to CA1 synapses, NRG1 effects are activity dependent and mediated via modulation of synaptic AMPARs.

Glioblastoma (GBM) is often resistant to conventional and targeted therapeutics. ErbB2 Receptor Tyrosine Kinase 4 (ERBB4) is expressed throughout normal brain and is an oncogene in several pediatric brain cancers; therefore, we investigated ERBB4 as a prognostic marker and therapeutic target in GBM. Using RT-qPCR, we quantified mRNA encoding total ERBB4 and known ERBB4 variants in GBM and non-neoplastic normal brain (NNB) samples. Using immunohistochemistry, we characterized the localization of total and phosphorylated ERBB4 (p-ERBB4) and EGFR protein in archived GBM samples and assessed their association with patient survival. Furthermore, we evaluated the effect of ERBB4 phosphorylation on angiogenesis and tumorigenicity in GBM xenograft models. Total ERBB4 mRNA was significantly lower in GBM than NNB samples, with the juxtamembrane JM-a and cytoplasmic CYT-2 variants predominating. ERBB4 protein was ubiquitously expressed in GBM but was not associated with patient survival. However, high p-ERBB4 in 11% of archived GBM samples, independent of p-EGFR, was associated with shorter patient survival (12.0 ± 3.2 months) than was no p-ERBB4 (22.5 ± 9.5 months). Increased ERBB4 activation was also associated with increased proliferation, angiogenesis, tumorigenicity and reduced sensitivity to anti-EGFR treatment in xenograft models. Despite low ERBB4 mRNA in GBM, the functional effects of increased ERBB4 activation identify ERBB4 as a potential prognostic and therapeutic target.

Epithelial-mesenchymal transition (EMT) is an important biological process that is characteristic of malignant tumor cells with metastatic potential. We investigated the role of miR-551b in EMT and metastasis in gastric cancer (GC). We found that low miR-551b levels were associated with EMT, metastasis and a poor prognosis in GC patients. Further, two GC cell lines, MNK45 and SGC7901, exhibited lower miR-551b levels than the GES normal stomach cell line. Exposing MNK45 and SGC7901 cells to TGF-β1 resulted in cell morphology changes characteristic of EMT, which was confirmed by Western blot analysis demonstrating low E-Cadherin and high N-Cadherin and Vimentin levels. Treatment with miR-551b mimics inhibited these EMT changes as well as Transwell migration and invasiveness. We identified ERBB4 as a potential target of miR-551b based on patient data from the TCGA. ERBB4 was upregulated in GC specimens, and its high expression correlated with a poor prognosis of GC patients. Dual luciferase assays revealed that miR-551b directly inhibited ERBB4 by binding to its 3'UTR. Moreover, treatment with miR-551b mimics or the ERBB4 inhibitor AST-1306 inhibited EMT in the GC cell lines. Finally, nude mice xenografted with GC cancer cell lines expressing miR-551b mimics exhibited smaller tumors and longer survival than mice engrafted with control GC cancer cells. These data indicate that miR-551b inhibits EMT and metastasis in GC by inhibiting ERBB4. miR-551b and ERBB4 are thus potential therapeutic targets for the treatment of GC.

Neuregulin-1 (NRG-1) is an endothelium-derived growth factor with cardioprotective and antiatherosclerotic properties and is currently being tested in clinical trials as a treatment for systolic heart failure. In clinical practice, heart failure often coexists with renal failure, sharing an overlapping pathophysiological background. In this study, we hypothesized that NRG-1 might protect against cardiomyopathy, atherosclerosis, and nephropathy within one disease process. We tested this hypothesis in a hypercholesterolemic apolipoprotein E-deficient (apoE(-/-)) type 1 diabetes mouse model prone to the development of cardiomyopathy, atherosclerosis, and nephropathy and compared the effects of NRG-1 with insulin. Upon onset of hyperglycemia induced by streptozotocin, apoE(-/-)mice were treated with vehicle, insulin, or recombinant human (rh)NRG-1 for 14 wk and were compared with nondiabetic apoE(-/-)littermates. Vehicle-treated diabetic apoE(-/-)mice developed left ventricular (LV) dilatation and dysfunction, dense atherosclerotic plaques, and signs of nephropathy. Nephropathy was characterized by abnormalities including hyperfiltration, albuminuria, increased urinary neutrophil gelatinase-associated lipocalin (NGAL), upregulation of renal fibrotic markers, and glomerulosclerosis. rhNRG-1 treatment induced systemic activation of ErbB2 and ErbB4 receptors in both heart and kidneys and prevented LV dilatation, improved LV contractile function, and reduced atherosclerotic plaque size. rhNRG-1 also significantly reduced albuminuria, NGALuria, glomerular fibrosis, and expression of fibrotic markers. Regarding the renal effects of rhNRG-1, further analysis showed that rhNRG-1 inhibited collagen synthesis of glomerular mesangial cells in vitro but did not affect AngII-induced vasoconstriction of glomerular arterioles. In conclusion, systemic administration of rhNRG-1 in hypercholesterolemic type 1 diabetic mice simultaneously protects against complications in the heart, arteries and kidneys.

Neural circuits in the cerebral cortex consist of excitatory pyramidal cells and inhibitory interneurons. These two main classes of cortical neurons follow largely different genetic programs, yet they assemble into highly specialized circuits during development following a very precise choreography. Previous studies have shown that signals produced by pyramidal cells influence the migration of cortical interneurons, but the molecular nature of these factors has remained elusive. Here, we identified Neuregulin 3 (Nrg3) as a chemoattractive factor expressed by developing pyramidal cells that guides the allocation of cortical interneurons in the developing cortical plate. Gain- and loss-of-function approaches reveal that Nrg3 modulates the migration of interneurons into the cortical plate in a process that is dependent on the tyrosine kinase receptor ErbB4. Perturbation of Nrg3 signaling in conditional mutants leads to abnormal lamination of cortical interneurons. Nrg3 is therefore a critical mediator in the assembly of cortical inhibitory circuits.

The newly discovered transactivation function of ErbB4 receptor tyrosine kinase is believed to be mediated by virtue of the ability of its proteolytically-cleaved intracellular domain (ICD) to physically associate with YAP2 transcriptional regulator. In an effort to unearth the molecular basis of YAP2-ErbB4 interaction, we have conducted a detailed biophysical analysis of the binding of WW domains of YAP2 to PPXY motifs located within the ICD of ErbB4. Our data show that the WW1 domain of YAP2 binds to PPXY motifs within the ICD in a differential manner and that this behavior is by and large replicated by the WW2 domain. Remarkably, while both WW domains absolutely require the integrity of the PPXY consensus sequence, non-consensus residues within and flanking this motif do not appear to be critical for binding. In spite of this shared mode of binding, the WW domains of YAP2 display distinct conformational dynamics in complex with PPXY motifs derived from ErbB4. Collectively, our study lends new insights into the molecular basis of a key protein-protein interaction involved in a diverse array of cellular processes.

Determining the molecular mechanism of human embryo implantation is an extremely challenging task due to the limitation of materials and significant differences in this process among mammalian species. Trophinin has been identified as an apical cell adhesion molecule with potential involvement in human embryo implantation. We found that trophinin-mediated cell adhesion triggers signal transduction in human trophoblastic cells for proliferation and invasion, implicating in trophectoderm cell activation for placental formation. Prior to cell adhesion trophinin arrests ErbB4 by binding through bystin, which prevents ErbB4 from activation. Trophinin-mediated cell adhesion causes dissociation of bystin from trophinin, freeing ErbB4 from arrest and enabling tyrosine phosphorylation. Therefore trophinin functions as an adhesion molecule on the cell surface and as a molecular switch for trophoblast activation in the cytoplasm.

It has been reported that specific environmental influences during the postpartum period might contribute to the development of schizophrenia (SZ). Administration of MK801 during early development led to persistent brain pathology. Glutamate decarboxylase 1 (GAD67) and parvalbumin (PV), and neuregulin 1 (NRG1)/ErbB4 signaling were closely associated with SZ pathology. We postulated therefore that NMDA receptor antagonists exposure during the postpartum period may be associated with expression dysregulation of some of the SZ candidate proteins. To test this, we used mouse primary hippocampal neurons and neonatal male mice treated with the NMDA receptor antagonist, MK801 at postnatal day 4 (P4) or P7, followed by the treatments of antipsychotic drugs (i.e., olanzapine, risperidone, and haloperidol). The expressions of GAD67, PV, NRG1, and ErbB4 in in vitro and in vivo SZ models were detected with Western blot analysis and immunohistochemistry, respectively. Behavioral tests (locomotion activity, social interaction, novel object recognition and prepulse inhibition) were measured. We found MK801 decreased the expression of GAD67, PV, NRG1 and ErbB4, and induced obvious behavioral alterations, while antipsychotics reversed these alterations. These results suggest that exposure to the NMDA receptor antagonist in early development may lead to long-lasting influence on the expression of specific proteins, such as GAD67, PV, NRG1, and ErbB4. Moreover, our results suggest that rescue of the activation of the NRG1/ErbB4 signaling pathway may be one of the mechanisms by which antipsychotic drugs have an antipsychotic effect.

We characterized the SLC3A2-NRG1 fusion gene in non-small cell lung cancer (NSCLC) and established an effective therapy for patients with SLC3A2-NRG1 fusion-positive cancer. The SLC3A2-NRG1 fusion product was composed of the SLC3A2 transmembrane domain and the EGF-like domain of the neuregulin 1 (NRG1) protein. The NRG1 family is classified as a ligand of the ERBB family. We identified ERBB3 and ERBB4 in the ERBB family as binding partners of the SLC3A2-NRG1 fusion protein via ligand and receptor binding assays. We confirmed that SLC3A2-NRG1 increased formation of a heterocomplex of ERBB3 with ERBB2. Activation of the ERBB2-ERBB3 heterocomplex by SLC3A2-NRG1 increased colony formation and tumor growth through PI3K-AKT and MAP kinase. The specific siRNAs for ERBB2 and ERBB3, pertuzumab, lumretuzumab, and afatinib all decreased ERBB2-ERBB3 heterocomplex formation, phosphorylation of each protein, and their downstream signaling. In addition, single treatment with pertuzumab, lumretuzumab, or afatinib decreased tumor volume and weight, whereas combination treatment with these drugs and taxol enhanced generation of cleaved caspase 3, PARP, and TUNEL-positive cells compared with each single treatment. Thus, the SLC3A2-NRG1 fusion gene plays an important role in lung cancer cell proliferation and tumor growth by promoting generation of the ERBB2-ERBB3 heterocomplex, its phosphorylation, and activation of the PI3K/ERK/mTOR signaling pathway. Inhibition of either ERBB2 or ERBB3 alone did not completely shut down downstream signaling of ERBB2 and ERBB3; however, inhibition of both ERBB2 and ERBB3 blocked downstream signaling activated by SLC3A2-NRG1 fusion. ERBB2 and ERBB3 might be promising targets for treatment of SLC3A2-NRG1-positive tumors. Mol Cancer Ther; 17(9); 2024-33. ©2018 AACR.

Pazopanib is US FDA approved for the treatment of advanced soft tissue sarcomas. All patients with this disease ultimately develop resistance to therapy. Mechanisms of resistance include activation of the mTOR, histone deacetylase (HDAC), MAPK, and ERBB4 pathways. We hypothesized that combining pazopanib with other targeted agents inhibiting these pathways would increase response rates. We retrospectively evaluated the safety and efficacy of pazopanib plus vorinostat, everolimus, lapatinib or trastuzumab, and MEK inhibitor in patients with advanced sarcoma. The Cancer Geneome Atlas (TCGA) data was analyzed for HDAC, PI3K, HER2, and MAPK/RAS/RAF gene alterations from sarcoma TCGA. Of the 44 advanced sarcoma patients in these trials, 27 (61%) were male; 18 (41%) had bone sarcoma, and 26 (59%) had soft tissue sarcoma. Best response was partial response (PR) in four patients [(overall response rate (ORR) = 9%, 95% confidence interval [CI] 3% to 22%)]. The median progression-free survival (PFS) for all patients was 9.6 weeks (95% CI 8.0 to 15.7 weeks). Analysis of TCGA data revealed HDAC, PI3K, HER2, and MAPK/RAS/RAF gene alterations in 112/243 (46%) of patients predominantly HDAC1-11 (41%) alterations. Pazopanib combinations did demonstrate safety in combination with other agents. TCGA data suggests further evaluation of epigenetic pathway inhibitors in sarcoma.

The neuregulin (NRG) family of epidermal growth factor-related proteins is composed of a wide variety of soluble and membrane-bound proteins that exert their effects via the tyrosine kinase receptors ErbB2-ErbB4. In the nervous system, the functions of NRG1 are essential for peripheral myelination, the establishment and maintenance of neuromuscular and sensorimotor systems and the plasticity of cortical neuronal circuits. In the present study, we report that an intracerebroventricular infusion of NRG1 attenuated cognitive impairments in 13-month-old Tg2576 mice, an animal model of Alzheimer's disease (AD). In addition, according to Golgi-Cox staining, NRG1 rescued the reduction in the number of dendritic spines detected in the brains of Tg2576 mice compared with vehicle (PBS)-infused mice. This result was also corroborated in vitro as NRG1 attenuated the oligomeric amyloid beta peptide(1-42) (Aβ(1-42))-induced decrease in dendritic spine density in rat primary hippocampal neuron cultures. NRG1 also alleviated the decrease in neural differentiation induced by oligomeric Aβ(1-42) in mouse fetal neural stem cells. Collectively, these results suggest that NRG1 has a therapeutic potential for AD by alleviating the reductions in dendritic spine density and neurogenesis found in AD brains.

The EGF receptor (EGFR) family comprises four homologs in humans collectively known as the ErbB or HER proteins. ErbB proteins are receptor tyrosine kinases that become activated when ligands bind to their extracellular regions and promote formation of specific homo- and heterodimers with enhanced tyrosine kinase activity. An essential feature of ErbB activation is formation of an asymmetric kinase dimer in which the C-terminal lobe of one kinase serves as the activator or donor kinase by binding the N-terminal lobe of a receiver or acceptor kinase and stabilizing its active conformation. ErbB extracellular regions are also thought to form active asymmetric dimers in which only one subunit binds ligand. The observation that the unliganded ErbB2 kinase preferentially serves as the activator kinase when paired with EGFR/ErbB1 implied that extracellular asymmetry in ErbB proteins might be coupled to intracellular asymmetry with unliganded partners favoring the activator kinase position. Using cell-based stimulation assays and chimeric ErbB proteins, we show that extracellular asymmetry is not coupled to intracellular asymmetry and that ErbB intracellular regions are sufficient to determine relative kinase activator-receiver orientation. We further show a hierarchy of activator-receiver preferences among ErbB proteins, with EGFR/ErbB1 being the strongest receiver, followed by ErbB2 and then ErbB4, and that cis-phosphorylation of EGFR and ErbB2 appears to be negligible. This hierarchy shapes the nature of signaling responses to different ligands in cells expressing multiple ErbB proteins.

Lung cancer is the leading cause of cancer related deaths worldwide and mutation activating KRAS is one of the most frequent mutations found in lung adenocarcinoma. Identifying regulators of KRAS may aid in the development of therapies to treat this disease. The mitogen-induced gene 6, MIG-6, is a small adaptor protein modulating signaling in cells to regulate the growth and differentiation in multiple tissues. Here, we investigated the role of Mig-6 in regulating adenocarcinoma progression in the lungs of genetically engineered mice with activation of Kras.

Using the CCSPCre mouse to specifically activate expression of the oncogenic KrasG12D in Club cells, we investigated the expression of Mig-6 in CCSPCreKrasG12D-induced lung tumors. To determine the role of Mig-6 in KrasG12D-induced lung tumorigenesis, Mig-6 was conditionally ablated in the Club cells by breeding Mig6f/f mice to CCSPCreKrasG12D mice, yielding CCSPCreMig-6d/dKrasG12D mice (Mig-6d/dKrasG12D).

We found that Mig-6 expression is decreased in CCSPCreKrasG12D-induced lung tumors. Ablation of Mig-6 in the KrasG12D background led to enhanced tumorigenesis and reduced life expectancy. During tumor progression, there was increased airway hyperplasia, a heightened inflammatory response, reduced apoptosis in KrasG12D mouse lungs, and an increase of total and phosphorylated ERBB4 protein levels. Mechanistically, Mig-6 deficiency attenuates the cell apoptosis of lung tumor expressing KRASG12D partially through activating the ErbB4 pathway.

In summary, Mig-6 deficiency promotes the development of KrasG12D-induced lung adenoma through reducing the cell apoptosis in KrasG12D mouse lungs partially by activating the ErbB4 pathway.

Each member of the epidermal growth factor receptor (EGFR) family plays a key role in normal development, homeostasis, and a variety of pathophysiological conditions, most notably in cancer. According to the prevailing dogma, these four receptor tyrosine kinases (RTKs; EGFR, ERBB2, ERBB3, and ERBB4) function exclusively through the formation of homodimers and heterodimers within the EGFR family. These combinatorial receptor interactions are known to generate increased interactome diversity and therefore influence signaling output, subcellular localization and function of the heterodimer. This molecular plasticity is also thought to play a role in the development of resistance toward targeted cancer therapies aimed at these known oncogenes. Interestingly, many studies now challenge this dogma and suggest that the potential for EGFR family receptors to interact with more distantly related RTKs is much greater than currently appreciated. Here we discuss how the promiscuity of these oncogenic receptors may lead to the formation of many unexpected receptor pairings and the significant implications for the efficiency of many targeted cancer therapies.

Hirschsprung disease (HSCR) is a complex birth defect characterized by the lack of ganglion cells along a variable length of the distal intestine. A large proportion of HSCR patients remain genetically unexplained. We applied whole-genome sequencing (WGS) on 9 trios where the probands are sporadically affected with the most severe form of the disorder and harbor no coding sequence variants affecting the function of known HSCR genes. We found de novo protein-altering variants in three intolerant to change genes-CCT2, VASH1, and CYP26A1-for which a plausible link with the enteric nervous system (ENS) exists. De novo single-nucleotide and indel variants were present in introns and non-coding neighboring regions of ENS-related genes, including NRG1 and ERBB4. Joint analysis with those inherited rare variants found under recessive and/or digenic models revealed both patient-unique and shared genetic features where rare variants were found to be enriched in the extracellular matrix-receptor (ECM-receptor) pathway (p = 3.4 × 10-11). Delineation of the genetic profile of each patient might help finding common grounds that could lead to the discovery of shared molecules that could be used as drug targets for the currently ongoing cell therapy effort which aims at providing an alternative to the surgical treatment.

Therapeutic effects of skeletal myoblast transplantation into the myocardium are mediated via paracrine factors. We investigated the ability of myoblast-derived soluble mediators to protect cardiomyocytes from oxidative stress. Fetal rat cardiac cells were treated with conditioned medium from cultures of myoblasts or cardiac fibroblasts, and oxidative stress was induced with H2O2. Myoblast-derived factors effectively prevented oxidative stress-induced cardiac cell death and loss of mitochondrial membrane potential. This protective effect was mediated via epidermal growth factor (EGF) receptor and c-Met signaling, and mimicked by neuregulin 1 but not EGF. Microarray analysis of cardiac cells treated with myoblast versus cardiac fibroblast-derived mediators revealed differential regulation of genes associated with antioxidative effects: cystathionine-γ-lyase (cst), xanthine oxidase, and thioredoxin-interacting protein as well as tribbles homolog 3 (trib3). Cardiac cell pretreatment with tunicamycin, an inducer of trib3, also protected them against H2O2-induced cell death. Epicardial transplantation of myoblast sheets in a rat model of acute myocardial infarction was used to evaluate the expression of CST and trib3 as markers of myoblasts' paracrine effect in vivo. Myoblast sheets induced expression of the CST as well as trib3 in infarcted myocardium. CST localized around blood vessels, suggesting smooth muscle cell localization. Our results provide a deeper molecular insight into the therapeutic mechanisms of myoblast-derived paracrine signaling in cardiac cells and suggest that myoblast transplantation therapy may prevent oxidative stress-induced cardiac deterioration and progression of heart failure.

The ERBB1-ERBB4 receptors belong to a family of receptor tyrosine kinases that trigger a network of signaling pathways after ligand binding, thereby regulating cellular growth, differentiation and development. Ligand-induced signaling through ERBB1, also known as EGFR, is attenuated by the clathrin-dependent receptor-mediated endocytosis and RING E3-ligase Cbl-mediated receptor ubiquitination, which is followed by incorporation into multi-vesicular bodies (MVBs) and subsequent degradation in lysosomes. Before incorporation into MVBs, the EGFR is deubiquitinated by Usp8. We previously demonstrated that Usp8 is tyrosine phosphorylated in an EGFR- and SRC-kinase dependent manner. In the present study we show that overexpression of constitutively active SRC enhances constitutive and ligand-induced Usp8 tyrosine phosphorylation. We also show that enhanced endosomal recycling of the EGFR induced by TGFα stimulation is associated with decreased Usp8 tyrosine phosphorylation. We therefore hypothesize that tyrosine phosphorylation of Usp8 could regulate the function of Usp8. To identify Usp8 tyrosine phosphorylation site(s), we used Usp8 deletion constructs, site-directed mutagenesis of nine individual Usp8 tyrosine residues and mass spectrometry (MS) analysis. Our results demonstrate that the MIT-domain is necessary for ligand-induced tyrosine phosphorylation of Usp8 1-504. However, mutation of three MIT domain tyrosine residues did not abolish Usp8 tyrosine phosphorylation. Similar results were obtained upon mutation of six exposed tyrosine residues in the Rhod domain and linker region. Repeated MS analysis of both Usp8 WT and C748A mutants readily detected serine phosphorylation, including the S680 14-3-3 binding site, but did not reveal any phospho-tyrosine residues. Notably, mutation of the tyrosine residue in the Usp8 14-3-3 binding motif (Y679) did not abolish phosphoserine-dependent binding of 14-3-3 to Usp8. Our findings are most consistent with the model that MIT domain-dependent recruitment of Usp8 to endosomal membranes is important for low stoichiometry SRC-mediated tyrosine phosphorylation of multiple Usp8 tyrosines. Our findings demonstrate that Usp8 is a target for the post-translational serine and tyrosine phosphorylation, most likely characterized by low abundant tyrosine phosphorylation on multiple residues, and high abundant serine phosphorylation on several residues.

To identify molecular alterations that contribute to vulvar cancer pathogenesis with the intent of identifying molecular targets for treatment.

After retrospective analysis of a database of molecularly-profiled gynecologic cancer patients, 149 vulvar cancer patients were included and tested centrally at a CLIA laboratory (Caris Life Sciences, Phoenix, AZ). Tests included one or more of the following: gene sequencing (Sanger or next generation sequencing [NGS]), protein expression (immunohistochemistry [IHC]), and gene amplification (C/FISH). A Fisher's exact test was used when indicated with a p-value≤0.05 indicating significance.

Median age was 65. 85% had squamous cell carcinoma (SCC) and 15% adenocarcinoma (ADC) histologies. 46% had metastatic (Stage IV) disease. Targeted hot-spot sequencing identified variants in the following genes: TP53 (33%), PIK3CA/BRCA2 (8%, 10%, respectively), HRAS/FBXW7 (5%, 4%, respectively) and ERBB4/GNAS (3%, 3% respectively). Mutations in AKT1, ATM, FGFR2, KRAS, NRAS (n=1, respectively) and BRAF (n=2) also occurred. Specific protein changes for targetable genes included clinically pathogenic mutations commonly found in other cancers (e.g. PIK3CA: exon 9 [E545K], RAS: G13D, Q61L, BRCA2: S1667X, BRAF: R443T, FBXW7: E471fs, etc.). Drug targets identified by IHC and ISH methodologies include cMET (32% IHC, 2% ISH), PDL1 (18%), PTEN loss (56%), HER2 (4% IHC, 2% ISH) and hormone receptors (AR, 4%; ER, 11%; PR, 4%). Comparisons between SCC and ADC identified differential rates for AR, ER, HER2 and GNAS with an increased presence in ADC (p-values all <0.05).

Molecularly-guided precision medicine could provide vulvar cancer patients alternative, targeted treatment options.

Heart failure is a complex syndrome whose phenotypic presentation and disease progression depends on a complex network of adaptive and maladaptive responses. One of these responses is the endothelial release of NRG (neuregulin)-1-a paracrine growth factor activating ErbB2 (erythroblastic leukemia viral oncogene homolog B2), ErbB3, and ErbB4 receptor tyrosine kinases on various targets cells. NRG-1 features a multitasking profile tuning regenerative, inflammatory, fibrotic, and metabolic processes. Here, we review the activities of NRG-1 on different cell types and organs and their implication for heart failure progression and its comorbidities. Although, in general, effects of NRG-1 in heart failure are compensatory and beneficial, translation into therapies remains unaccomplished both because of the complexity of the underlying pathways and because of the challenges in the development of therapeutics (proteins, peptides, small molecules, and RNA-based therapies) for tyrosine kinase receptors. Here, we give an overview of the complexity to be faced and how it may be tackled.

BACKGROUND

A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium fluctuations play central roles in neuronal motility. Stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation. In this work we analyzed the potential interactions between the NRG1/ErbB4 system and NMDARs in the ST14A migratory process as well as its calcium dependence.

RESULTS

RT-PCR studies have shown that both native ST14A cells (non-expressing ErbB4), as well as ErbB4-transfected cells express low levels of a restricted number of NMDAR subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form Ca(2+) channels characterized by low Mg(2+)-sensitivity and low Ca(2+)-permeability, generating small, long-lasting currents. Ca(2+)-imaging experiments showed slow [Ca(2+)](i) increases in 45% of the cells following 8 μM NMDA stimulation. Basal migration of ErbB4-transfected ST14A cells was unaffected by 18 hrs NMDA incubation. However, over the same incubation time, NMDA was able to significantly enhance NRG1-induced migration. Pre-incubation with the intracellular calcium chelator BAPTA-AM reduced both NRG1- and NRG1/NMDA-stimulated migration, suggesting the involvement of Ca(2+) in these processes. NRG1 stimulation of ErbB4-transfected ST14A cells induced a sustained, long-lasting increase in [Ca(2+)](i), in 99% of the cells. These intracellular Ca(2+) signals could be ascribed to both release from intracellular stores and influx from the extracellular medium trough a mechanism of store-operated calcium entry (SOCE). Short-time co-incubation of NMDA and NRG1 did not substantially modify the NRG1-induced intracellular calcium signals.

CONCLUSIONS

In summary, NRG1 stimulation of the ErbB4 receptor exerts a sustained [Ca(2+)](i) increase in ST14A neural progenitors; NRG1-induced migration is Ca(2+)-dependent and can be positively modulated by activation of the NMDA receptor.