Schizophrenia is one of the most common and complex neuropsychiatric disorders, which is contributed both by genetic and environmental exposures. Recently, it is shown that NRG1-mediated ErbB4 signalling regulates many important cellular and molecular processes such as cellular growth, differentiation and death, particularly in myelin-producing cells, glia and neurons. Recent association studies have revealed genomic regions of NRG1 and ERBB4, which are significantly associated with risk of developing schizophrenia; however, inconsistencies exist in terms of validation of findings between distinct populations. In this study, we aim to validate the previously identified regions and to discover novel haplotypes of NRG1 and ERBB4 using logistic regression models and Haploview analyses in three independent datasets from GWAS conducted on European subjects, namely, CATIE, GAIN and nonGAIN. We identified a significant 6-kb block in ERBB4 between chromosome locations 212,156,823 and 212,162,848 in CATIE and GAIN datasets (p = 0.0206 and 0.0095, respectively). In NRG1, a significant 25-kb block, between 32,291,552 and 32,317,192, was associated with risk of schizophrenia in all CATIE, GAIN, and nonGAIN datasets (p = 0.0005, 0.0589, and 0.0143, respectively). Fine mapping and FastSNP analysis of genetic variation located within significantly associated regions proved the presence of binding sites for several transcription factors such as SRY, SOX5, CEPB, and ETS1. In this study, we have discovered and validated haplotypes of ERBB4 and NRG1 in three independent European populations. These findings suggest that these haplotypes play an important role in the development of schizophrenia by affecting transcription factor binding affinity.

The ErbB family of receptor tyrosine kinases includes the epidermal growth factor receptor (EGFR), p185/neu/c-erbB2, ErbB3, and ErbB4. Many of these receptors are overexpressed or amplified in various forms of cancers. Previous studies have indicated that activation of erbB molecules contributes to malignant transformation both by promoting cell proliferation through the mitogen-activated protein kinase (MAP kinase) signaling pathway and by preventing apoptosis through the Phosphoinositide 3 kinase (PI-3 kinase) pathway. Disabling erbB receptors converts malignant cells that were resistant to cell death caused by irradiation to cells that are sensitive to apoptosis. Here, we report that an activated form of EGFR can elevate the levels of Survivin, a member of the Inhibitor of Apoptosis Protein (IAP) family implicated in mitotic checkpoint control. Conversely, inactivation of the ErbB receptors reduces the expression levels of Survivin. Furthermore, we found that upregulation of Survivin by EGFR is dependent on the PI-3 kinase pathway but not on the MAP kinase pathway. Indeed, inhibition of PI-3 kinase can diminish Survivin at both the mRNA and the protein levels. Combined with previous findings that Survivin plays a role in control of chromosome segregation and that it is overexpressed in various cancers, our results suggest that EGFR may cause transformation by directly affecting mitosis and increasing chromosome instability.

Variations in the signalling NRG1-ErbB4 pathway have been associated with genetic susceptibility for both bipolar disorder and schizophrenia, although the underlying neural mechanisms are still uncertain. Reduced integrity of the anterior limb of the internal capsule (ALIC) has been found in association with risk-associated genetic variation in the 5' region of the NRG1 gene. We hypothesised that variation in the gene encoding the NRG1 receptor, ErbB4, would also be associated with reduced ALIC integrity and with cognitive impairments characteristic of individuals with bipolar disorder and schizophrenia. Using diffusion tensor imaging (DTI), we examined the white matter integrity associations of the ErbB4 polymorphism rs4673628, which resides within intron 12 of the gene encoding ErbB4, in 36 healthy individuals. We also sought to clarify the cognitive effects of any findings. We found that genetic variation at the rs4673628 locus in the ErbB4 gene was significantly associated with ALIC white matter integrity which was also significantly and positively associated with mnemonic function. These findings provide further evidence to support a key role of NRG1-ErbB4 signalling in the pathophysiology of major mental disorders.

The gamma-secretase complex catalyzes intramembrane proteolysis of a number of transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and E-cadherin. gamma-Secretase is known to contain four major protein constituents: presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane proteins. There is increasing evidence that the formation of the complex and the stability of the individual components are tightly controlled in the cell, assuring correct composition of functional complexes. In this report, we investigate the topology, localization, and mechanism for destabilization of Pen-2 in relation to PS function. We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature gamma-secretase complexes reside. PS deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2, which adopts a hairpin structure with the N and C termini facing the luminal space, is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the gamma-secretase complex leads to degradation of Pen-2 through the ER-associated degradation-proteasome pathway.

Transient elevation of intracellular calcium (Ca2+(i)) by various means accelerates murine preimplantation development and trophoblast differentiation. Several G-protein-coupled receptors (GPCRs), including the lysophosphatidic acid (LPA) receptor (LPAR), induce Ca2+(i) transients and transactivate the EGF receptor (ErbB1) through mobilization of EGF family members, including heparin-binding EGF-like growth factor (HB-EGF). Because HB-EGF accelerates blastocyst differentiation in vitro, we examined whether crosstalk between LPA and HB-EGF regulates peri-implantation development. During mouse blastocyst differentiation, embryos expressed LPAR1 mRNA constitutively, LPAR2 only in late stage blastocysts and no LPAR3. Consistent with a mechanism based on Ca2+(i) signaling, LPA rapidly accelerated the rate of trophoblast outgrowth, an index of blastocyst differentiation, and chelation of Ca2+(i) with BAPTA-AM blocked LPA stimulation. Interfering with HB-EGF signaling through ErbB1 or ErbB4 also attenuated LPA stimulation. We established that mouse blastocysts indeed express HB-EGF and that LPA induces the transient accumulation of HB-EGF on the embryo surface, which was blocked by treatment with either BAPTA-AM or the protein trafficking inhibitor, brefeldin A. We conclude that LPA accelerates blastocyst differentiation through its ability to induce Ca2+(i) transients and HB-EGF autocrine signaling. Transactivation of ErbB1 or ErbB4 by HB-EGF could represent a convergent signaling pathway accessed in the trophoblast by stimuli that mobilize Ca2+(i).

Receptor tyrosine kinases (RTKs) and their downstream signalling pathways have long been hypothesized to play key roles in melanoma development. A decade ago, evidence was derived largely from animal models, RTK expression studies and detection of activated RAS isoforms in a small fraction of melanomas. Predictions that overexpression of specific RTKs implied increased kinase activity and that some RTKs would show activating mutations in melanoma were largely untested. However, technological advances including rapid gene sequencing, siRNA methods and phospho-RTK arrays now give a more complete picture. Mutated forms of RTK genes including KIT, ERBB4, the EPH and FGFR families and others are known in melanoma. Additional over- or underexpressed RTKs and also protein tyrosine phosphatases (PTPs) have been reported, and activities measured. Complex interactions between RTKs and PTPs are implicated in the abnormal signalling driving aberrant growth and survival in malignant melanocytes, and indeed in normal melanocytic signalling including the response to ultraviolet radiation. Kinases are considered druggable targets, so characterization of global RTK activity in melanoma should assist the rational development of tyrosine kinase inhibitors for clinical use.

The ERBB4 receptor tyrosine kinase promotes colonocyte survival. Herein, we tested whether ERBB4's antiapoptotic signaling promotes transformation and colorectal tumorigenesis. ERBB4 alterations in a The Cancer Genome Atlas colorectal cancer (CRC) data set stratified survival, and in a combined Moffitt Cancer Center and Vanderbilt Medical Center CRC expression data set, ERBB4 message levels were increased at all tumor stages. Similarly, western blot and immunohistochemistry on additional CRC tissue banks showed elevated ERBB4 protein in tumors. ERBB4 was highly expressed in aggressive, dedifferentiated CRC cell lines, and its knockdown in LIM2405 cells reduced anchorage-independent colony formation. In nude mouse xenograft studies, ERBB4 alone was insufficient to induce tumor establishment of non-transformed mouse colonocytes, but its over-expression in cells harboring Apc(min) and v-Ha-Ras caused a doubling of tumor size. ERBB4-expressing xenografts displayed increased activation of survival pathways, including epidermal growth factor receptor and Akt phosphorylation and COX-2 expression, and decreased apoptotic signals. Finally, ERBB4 deletion from mouse intestinal epithelium impaired stem cell replication and in vitro enteroid establishment. In summary, we report that ERBB4 is over-expressed in human CRC, and in experimental systems enhances the survival and growth of cells driven by Ras and/or WNT signaling. Chronic ERBB4 over-expression in the context of, for example, inflammation may contribute to colorectal carcinogenesis. Tumors with high receptor levels are likely to have enhanced cell survival signaling through epidermal growth factor receptor, PI3K and COX-2. These results suggest ERBB4 as a novel therapeutic target in a subset of CRC.

Transforming growth factor-alpha (TGF-alpha) is abnormally expressed in autosomal recessive polycystic kidney disease (ARPKD). Tumor necrosis factor-alpha converting enzyme (TACE), a metalloproteinase, mediates TGF-alpha processing. In this study, we sought to determine whether TGF-alpha was an absolute requirement for renal cystogenesis and whether its absence would modulate disease severity or related growth factors/receptors expression. Bpk heterozygotes were bred with TGF-alpha null mice to produce cystic and noncystic offspring with or without TGF-alpha. Assessments included kidney weight (KW), body weight (BW), blood urea nitrogen (BUN), and kidney and liver immunohistology. Western analysis assessed kidney expression of amphiregulin (AR), epidermal growth factor (EGF), heparin-binding EGF (HB-EGF), and their receptors, EGFR and ErbB4. A PCR-based methodology for genotyping bpk mice was also developed. No significant differences in KW, BW, KW/BW%, or BUN were seen in cystic mice with versus without TGF-alpha. Cystic kidney disease and liver disease histology were similar. AR, EGF, HB-EGF, EGFR, and ErbB4 were abnormally expressed to an equal degree in kidneys of mice with versus without TGF-alpha. Although previous data suggest a critical role of TGF-alpha in murine PKD, these data show that TGF-alpha is not required for renal cyst formation or kidney or liver disease progression. We speculate that the therapeutic effect of WTACE2 could have been due to effects on several TACE targets, including TGF-alpha, AR, and ErbB4, as well as metalloproteinases other than TACE.

Smoking is a major risk factor for several somatic diseases and is also emerging as a causal factor for neuropsychiatric disorders. Genome-wide association (GWA) and candidate gene studies for smoking behavior and nicotine dependence (ND) have disclosed too few predisposing variants to account for the high estimated heritability. Previous large-scale GWA studies have had very limited phenotypic definitions of relevance to smoking-related behavior, which has likely impeded the discovery of genetic effects. We performed GWA analyses on 1114 adult twins ascertained for ever smoking from the population-based Finnish Twin Cohort study. The availability of 17 smoking-related phenotypes allowed us to comprehensively portray the dimensions of smoking behavior, clustered into the domains of smoking initiation, amount smoked and ND. Our results highlight a locus on 16p12.3, with several single-nucleotide polymorphisms (SNPs) in the vicinity of CLEC19A showing association (P<1 × 10(-6)) with smoking quantity. Interestingly, CLEC19A is located close to a previously reported attention-deficit hyperactivity disorder (ADHD) linkage locus and an evident link between ADHD and smoking has been established. Intriguing preliminary association (P<1 × 10(-5)) was detected between DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, 4th edition) ND diagnosis and several SNPs in ERBB4, coding for a Neuregulin receptor, on 2q33. The association between ERBB4 and DSM-IV ND diagnosis was replicated in an independent Australian sample. Recently, a significant increase in ErbB4 and Neuregulin 3 (Nrg3) expression was revealed following chronic nicotine exposure and withdrawal in mice and an association between NRG3 SNPs and smoking cessation success was detected in a clinical trial. ERBB4 has previously been associated with schizophrenia; further, it is located within an established schizophrenia linkage locus and within a linkage locus for a smoker phenotype identified in this sample. In conclusion, we disclose novel tentative evidence for the involvement of ERBB4 in ND, suggesting the involvement of the Neuregulin/ErbB signalling pathway in addictions and providing a plausible link between the high co-morbidity of schizophrenia and ND.

The metastatic process requires changes in tumor cell adhesion properties, cell motility and remodeling of the extracellular matrix. The erbB2 proto-oncogene is overexpressed in approximately 30% of breast cancers and is a major prognostic parameter when present in invasive disease. A ligand for the erbB2 receptor has not yet been identified but it can be activated by heterodimerization with heregulin (HRG)-stimulated erbB3 and erbB4 receptors. The HRGs are a family of polypeptide growth factors that have been shown to play a role in embryogenesis, tumor formation, growth and differentiation of breast cancer cells. The erbB3 and erbB4 receptors are involved in transregulation of erbB2 signaling. The work presented here suggests biological roles for HRG including regulation of the actin cytoskeleton and induction of motility and invasion in breast cancer cells. HRG-expressing breast cancer cell lines are characterized by low erbB receptor levels and a high invasive and metastatic index, while those which overexpress erbB2 demonstrate minimal invasive potential in vitro and are non-tumorigenic in vivo. Treatment of the highly tumorigenic and metastatic HRG-expressing breast cancer cell line MDA-MB-231 with an HRG-neutralizing antibody significantly inhibited proliferation in culture and motility in the Boyden chamber assay. Addition of exogenous HRG to non-invasive erbB2 overexpressing cells (SKBr-3) at low concentrations induced formation of pseudopodia, enhanced phagocytic activity and increased chemomigration and invasion in the Boyden chamber assay. The specificity of the chemomigration response to HRG is demonstrated by inhibition with the anti-HRG neutralizing antibody. These results suggest that either HRG can act as an autocrine or paracrine ligand to promote the invasive behavior of breast cancer cells in vitro or thus may enhance the metastatic process in vivo.

Neuregulin-1 (NRG1) and its receptor ErbB4 influence several processes of neurodevelopment, but the mechanisms regulating this signalling in the mature brain are not well known. DISC1 is a multifunctional scaffold protein that mediates many cellular processes. Here we present a functional relationship between DISC1 and NRG1-ErbB4 signalling in mature cortical interneurons. By cell type-specific gene modulation in vitro and in vivo including in a mutant DISC1 mouse model, we demonstrate that DISC1 inhibits NRG1-induced ErbB4 activation and signalling. This effect is likely mediated by competitive inhibition of binding of ErbB4 to PSD95. Finally, we show that interneuronal DISC1 affects NRG1-ErbB4-mediated phenotypes in the fast spiking interneuron-pyramidal neuron circuit. Post-mortem brain analyses and some genetic studies have reported interneuronal deficits and involvement of the DISC1, NRG1 and ErbB4 genes in schizophrenia, respectively. Our results suggest a mechanism by which cross-talk between DISC1 and NRG1-ErbB4 signalling may contribute to these deficits.

The epidermal growth factor receptor (EGFR) signaling pathways are altered in many cancers contributing to increased cell survival. These alterations are caused mainly through increased expression or mutation of EGFR family members EGFR, ErbB2, ErbB3, and ErbB4. These receptors have been successfully targeted for cancer therapy. Specifically, a monoclonal antibody against ErbB2, trastuzumab, and a tyrosine kinase inhibitor against EGFR, gefitinib, have improved the survival of breast and lung cancer patients. Unfortunately, cancer patients frequently become resistant to these inhibitors. This has led to investigating how EGFR can contribute to cell survival and how cancer cells can overcome inhibition of its signaling. Indeed, it is coming into focus that EGFR signaling goes beyond a single signal triggering cell proliferation and survival and is a sensor that regulates the cell's response to microenvironmental stresses such as hypoxia. It acts as a switch that modulates the ability of cancer cells to survive. Autophagy is a process of self-digestion that is inhibited by EGFR allowing cancer cells to survive under stresses that would normally cause death and become resistant to chemotherapy. Inhibiting EGFR signaling allows autophagy to contribute to cell death. This gives new opportunities to develop novel therapeutic strategies to treat cancers that rely on EGFR signaling networks and autophagy. In this review, we summarize the current understanding of EGFR family member regulation of autophagy in cancer cells and how new therapeutic strategies could be developed to overcome drug resistance.

Dermatologic adverse events (AEs) are frequently observed in patients receiving EGF receptor (EGFR; also known as ErbB1) tyrosine kinase inhibitor therapy. The impact of these AEs goes beyond cosmesis to the discomfort from itching, pain and secondary infections, all of which may significantly impact on patient well-being, adherence and clinical outcomes. Afatinib is a potent, irreversible, oral, ErbB family blocker, inhibiting EGFR (ErbB1), HER2 (ErbB2) and ErbB4 receptor kinases. It also inhibits transphosphorylation of ErbB3. Similar to EGFR inhibitors, dermatologic AEs have been frequently observed in patients treated with afatinib. Papulopustular (acneiform) rash, pruritus, xerosis, paronychia and alopecia will require patient education and proactive treatment interventions. This article summarizes current data on the dermatologic AEs associated with afatinib treatment across the clinical trial program, and provides strategies for their effective management.

ADAM10, overexpressed in tongue squamous cell carcinoma (TSCC), has been well documented for its role in tumor progression and metastasis. In the present study, we evaluated the inhibition effect of microRNAs (miRNAs) on the TSCC and identified that miR-140-5p could directly targets ADAM10 and inhibits the invasion and migration of TSCC cells. LAMC1, HDAC7 and PAX6, clustered into migration-related genes, were validated to be direct targets of miR-140-5p, while IGF1R and PSEN1 were not responsible to the regulation. Most intriguingly, ERBB4 was upregulated by miR-140-5p even though the interaction between ERBB4 3'UTR and miR-140-5p existed simultaneously. Meanwhile, ADAM10 is involved in the "positive" regulation of ERBB4 and negative regulation of PAX6 by miR-140-5p. Taken together, our results suggest that miR-140-5p play a role in TSCC cell migration and invasion, and two brand new relationships between miRNA and its targets emerged: (1) ADAM10 is not just a direct target of miR-140-5p, the repressed ADAM10 also helps to enhance the effect of miR-140-5p to other target genes: ERBB4 and PAX6; (2) ERBB4 is "positively" regulated by miR-140-5p.

Acinic cell carcinoma (ACC) of the breast is a rare form of triple-negative (that is, oestrogen receptor-negative, progesterone receptor-negative, HER2-negative) salivary gland-type tumour displaying serous acinar differentiation. Despite its triple-negative phenotype, breast ACCs are reported to have an indolent clinical behaviour. Here, we sought to define whether ACCs have a mutational repertoire distinct from that of other triple-negative breast cancers (TNBCs). DNA was extracted from microdissected formalin-fixed, paraffin-embedded sections of tumour and normal tissue from two pure and six mixed breast ACCs. Each tumour component of the mixed cases was microdissected separately. Tumour and normal samples were subjected to targeted capture massively parallel sequencing targeting all exons of 254 genes, including genes most frequently mutated in breast cancer and related to DNA repair. Selected somatic mutations were validated by targeted amplicon resequencing and Sanger sequencing. Akin to other forms of TNBC, the most frequently mutated gene found in breast ACCs was TP53 (one pure and six mixed cases). Additional somatic mutations affecting breast cancer-related genes found in ACCs included PIK3CA, MTOR, CTNNB1, BRCA1, ERBB4, ERBB3, INPP4B, and FGFR2. Copy number alteration analysis revealed complex patterns of gains and losses similar to those of common forms of TNBCs. Of the mixed cases analysed, identical somatic mutations were found in the acinic and the high-grade non-acinic components in two out of four cases analysed, providing evidence of their clonal relatedness. In conclusion, breast ACCs display the hallmark somatic genetic alterations found in high-grade forms of TNBC, including complex patterns of gene copy number alterations and recurrent TP53 mutations. Furthermore, we provide circumstantial genetic evidence to suggest that ACCs may constitute the substrate for the development of more aggressive forms of triple-negative disease.

BACKGROUND

Schizophrenia (SCZ) and type 2 diabetes mellitus (T2D) are both complex diseases. Accumulated studies indicate that schizophrenia patients are prone to present the type 2 diabetes symptoms, but the potential mechanisms behind their association remain unknown. Here we explored the pathogenetic association between SCZ and T2D based on pathway analysis and protein-protein interaction.

RESULTS

With sets of prioritized susceptibility genes for SCZ and T2D, we identified significant pathways (with adjusted p-value < 0.05) specific for SCZ or T2D and for both diseases based on pathway enrichment analysis. We also constructed a network to explore the crosstalk among those significant pathways. Our results revealed that some pathways are shared by both SCZ and T2D diseases through a number of susceptibility genes. With 382 unique susceptibility proteins for SCZ and T2D, we further built a protein-protein interaction network by extracting their nearest interacting neighbours. Among 2,104 retrieved proteins, 364 of them were found simultaneously interacted with susceptibility proteins of both SCZ and T2D, and proposed as new candidate risk factors for both diseases. Literature mining supported the potential association of partial new candidate proteins with both SCZ and T2D. Moreover, some proteins were hub proteins with high connectivity and interacted with multiple proteins involved in both diseases, implying their pleiotropic effects for the pathogenic association. Some of these hub proteins are the components of our identified enriched pathways, including calcium signaling, g-secretase mediated ErbB4 signaling, adipocytokine signaling, insulin signaling, AKT signaling and type II diabetes mellitus pathways. Through the integration of multiple lines of information, we proposed that those signaling pathways, which contain susceptibility genes for both diseases, could be the key pathways to bridge SCZ and T2D. AKT could be one of the important shared components and may play a pivotal role to link both of the pathogenetic processes.

CONCLUSIONS

Our study is the first network and pathway-based systematic analysis for SCZ and T2D, and provides the general pathway-based view of pathogenetic association between two diseases. Moreover, we identified a set of candidate genes potentially contributing to the linkage between these two diseases. This research offers new insights into the potential mechanisms underlying the co-occurrence of SCZ and T2D, and thus, could facilitate the inference of novel hypotheses for the co-morbidity of the two diseases. Some etiological factors that exert pleiotropic effects shared by the significant pathways of two diseases may have important implications for the diseases and could be therapeutic intervention targets.

Somatic mutations and copy number variation in the ERBB family are frequent in urothelial carcinoma (UC) and may represent viable therapeutic targets. We studied whether afatinib (an oral, irreversible inhibitor of the ErbB family) has activity in UC and if specific ERBB molecular alterations are associated with clinical response.

In this phase II trial, patients with metastatic platinum-refractory UC received afatinib 40 mg/day continuously until progression or intolerance. The primary end point was 3-month progression-free survival (PFS3). Prespecified tumor analysis for alterations in EGFR, HER2, ERBB3, and ERBB4 was conducted.

The first-stage enrollment goal of 23 patients was met. Patient demographic data included: 78% male, median age 67 years (range, 36 to 82 years), hemoglobin < 10 g/dL in 17%, liver metastases in 30%, median time from prior chemotherapy of 3.6 months, and Eastern Cooperative Oncology Group performance status ≤ 1 in 100%. No unexpected toxicities were observed; two patients required dose reduction for grade 3 fatigue and rash. Overall, five of 23 patients (21.7%) met PFS3 (two partial response, three stable disease). Notably, among the 21 tumors analyzed, five of six patients (83.3%) with HER2 and/or ERBB3 alterations achieved PFS3 (PFS = 10.3, 7.0, 6.9, 6.3, and 5.0 months, respectively) versus none of 15 patients without alterations (P < .001). Three of four patients with HER2 amplification and three of three patients with ERBB3 somatic mutations (G284R, V104M, and R103G) met PFS3. One patient with both HER2 amplification and ERBB3 mutation never progressed on therapy, but treatment was discontinued after 10.3 months as a result of depressed ejection fraction. The median time to progression/discontinuation was 6.6 months in patients with HER2/ERBB3 alterations versus 1.4 months in patients without alterations (P < .001).

Afatinib demonstrated significant activity in patients with platinum-refractory UC with HER2 or ERBB3 alterations. The potential contribution of ERBB3 to afatinib sensitivity is novel. Afatinib deserves further investigation in molecularly selected UC.

Despite the strongly held view that schizophrenia (SZ) shows substantial genetic heterogeneity, pathway heterogeneity, as seen in cancer where different pathways are affected in similar tumors, has not been explored. We explore this possibility in a case-only study of the neuregulin signaling pathway (NSP), which has been prominently implicated in SZ and for which there is detailed knowledge on the ligand- and receptor-processing steps through β- and γ-secretase cleavage. We hypothesize that more than one damaging variants in the NSP genes might be necessary to cause disease, leading to an apparent clustering of such variants in only the few patients with affected NSP. We analyze linkage and next-generation sequencing results for the genes encoding components of the pathway, including NRG1, NRG3, ERBB4, β-secretase and the γ-secretase complex. We find multiple independent examples of supporting evidence for this hypothesis: (i) increased linkage scores over NSP genes, (ii) multiple positive interlocus correlations of linkage scores across families suggesting each family is linked to either many or none of the genes, (iii) aggregation of predicted damaging variants in a subset of individuals and (iv) significant phenotypic differences of the subset of patients carrying such variants. Collectively, our data strongly support the hypothesis that the NSP is affected by multiple damaging variants in a subset of phenotypically distinct patients. On the basis of this, we propose a general model of pathway heterogeneity in SZ, which, in part, may explain its phenotypic variability and genetic complexity.

We have compared all available deduced protein sequences of the ErbB family of receptors and their ligands. Analysis of the aligned sequences of the receptors indicates that there are some differences in the receptors that are specific to invertebrates. In addition, comparison of the vertebrate ErbB receptors suggest that a gene duplication event generated two ancestral receptors, the ErbB3/ErbB4 precursor and the ErbB1/ErbB2 precursor. Subsequent gene duplications of these precursors generated the four receptors present in mammals. Analysis of the sequences for the known ligands of the ErbB receptors suggests that the vertebrate ligands segregate into the ErbB1 ligands and the ErbB3/ErbB4 ligands, paralleling the evolution of the receptors; however, it is difficult to ascertain any correlation between the invertebrate and the vertebrate ligands. Even though ErbB3 is kinase-impaired, there is significant conservation of the kinase domain within the vertebrate lineage (human, rat, and F. rubripes), suggesting some function for this domain other than kinase activity, such as mediating protein-protein interactions that are involved in receptor dimerization and/or activation of the kinase domain of the heterodimerization partner. To date, no ligand for ErbB2 has been identified, and comparison of the extracellular domains of ErbB2 reveals two regions that are not conserved across the mammalian species. These two regions of divergence align with sequences in ErbB1 that have been shown to be proximal to the amino-terminus and to the carboxyl-terminal region, respectively, of bound EGF. Further, one of these regions contains an insertion, relative to the other members of the mammalian ErbB family, which might affect the ligand binding site and provide a structural basis for this receptor's apparent inability to bind ligand independently.

Neuregulin 1 and its receptor ErbB4 are confirmed risk genes for schizophrenia, but the neuropathological alterations in NRG1-ErbB4 in schizophrenia are unclear. The present investigations therefore focused on determining lamina specific (ErbB4-pan) and quantitative (pan, JMa, JMb, CYT1 and CYT2) ErbB4 mRNA changes in the dorsolateral prefrontal cortex (DLPFC) in schizophrenia. We also determined which neuronal profiles are ErbB4 mRNA+ in the human DLPFC and the relationship between ErbB4 and interneuron marker mRNAs. In situ hybridisation and quantitative PCR measurements were performed to determine changes in ErbB4 splice variant mRNA levels in the DLPFC in schizophrenia (n = 37) compared to control (n = 37) subjects. Cortical neurons expressing ErbB4-pan were labelled with silver grain clusters. Correlations were performed between ErbB4 and interneuron mRNA levels. ErbB4-pan mRNA was significantly increased (layers I, II and V) in the DLPFC in schizophrenia. Silver grain clusters for ErbB4-pan were detected predominantly over small-medium neurons with low-no expression in the larger, paler, more triangular neuronal profiles. ErbB4-JMa mRNA expression was increased in schizophrenia. Somatostatin, neuropeptide Y and vasoactive intestinal peptide mRNAs negatively correlated with ErbB4-JMa mRNA in people with schizophrenia. Our findings demonstrate that ErbB4-pan laminar mRNA expression is elevated (layers I, II, V) in schizophrenia. At the cellular level, ErbB4-pan mRNA+ signal was detected predominantly in interneuron-like neurons. We provide evidence from this independent Australian postmortem cohort that ErbB4-JMa expression is elevated in schizophrenia and is linked to deficits in dendrite-targeting somatostatin, neuropeptide Y and vasoactive intestinal peptide interneurons.

Betacellulin (BTC) is a member of the epidermal growth factor (EGF) family, and it acts through EGF receptors. We asked whether BTC could be an angiogenic factor. Using human umbilical vein endothelial cells (HUVECs), we examined the effect of BTC on kinases and angiogenic processes. BTC induced ERK1/2 and Akt phosphorylation in a dose- and time-dependent manner. BTC induced phosphorylation of all three EGF receptors present on HUVECs: ErbB2, ErbB3, and ErbB4. Pretreatment with effective concentrations of ErbB1 inhibitor did not suppress BTC-induced kinase phosphorylation. BTC, EGF, VEGF (all at 10 ng/ml) produced similar increases in DNA synthesis. BTC, EGF, and VEGF all significantly increased endothelial cell migration. In addition, BTC promoted survival in a dose-dependent manner, and its effect was inhibited by pretreatment with PtdIns 3'-kinase inhibitor wortmannin or MEK1/2 inhibitor PD98059. Both BTC and EGF produced similar increases in tube formation in collagen gels. BTC-induced tube formation was suppressed by PD98059, wortmannin, and LY294002. In the mouse Matrigel plug assay, BTC (100 ng/ml) promoted neovessel formation, and its effect was suppressed by a combination of wortmannin and PD98059. Taken together, these data show that BTC exerts potent angiogenic activity through activation of EGF receptors, mitogen-activated protein kinase, and PtdIns 3'-kinase/Akt in endothelial cells.

Schizophrenia is a debilitating neurodevelopmental psychiatric disorder. Both the N-methyl-D-aspartate receptor (NMDAR) and neuregulin1 (NRG1) are key molecules involved in normal brain development that have been linked to schizophrenia pathology and aetiology. The NR2 proteins are critical structural and functional subunits of the NMDAR and are developmentally and spatially regulated. Altered NR2 gene and protein expression has been found in human post-mortem schizophrenia brain tissue together with changes in NRG1 and its receptor ErbB4. The NR2 subunits and ErbB4 share a common anchoring domain on the postsynaptic density and therefore a disruption to either of these molecules may influence the functioning of the other. It has been shown that NRG1 signalling can affect NMDAR levels and function, particularly phosphorylation of the NR2 subunits. However little is known about the possible effects of NMDAR dysfunction on NRG1 signalling, which is important with regards to schizophrenia aetiology as numerous risk factors for the disorder can alter NMDAR functioning during early brain development. This review focuses on the role of the NMDA receptor subunits and NRG1 signalling in schizophrenia and proposes a mechanism by which a disruption to the NMDAR, particularly via altering the balance of NR2 subunits during early development, could influence NRG1 signalling.

BACKGROUND

Metastasis is an important step in tumor progression leading to a disseminated and often incurable disease. First steps of metastasis include down-regulation of cell adhesion molecules, alteration of cell polarity and reorganization of cytoskeleton, modifications associated with enhanced migratory properties and resistance of tumor cells to anoikis. Such modifications resemble Epithelial to Mesenchymal Transition (EMT). In breast cancer CD146 expression is associated with poor prognosis and enhanced motility.

RESULTS

On 4 different human breast cancer cell lines, we modified CD146 expression either with shRNA technology in CD146 positive cells or with stable transfection of CD146 in negative cells. Modifications in morphology, growth and migration were evaluated. Using Q-RT-PCR, we analyzed the expression of different EMT markers. We demonstrate that high levels of CD146 are associated with loss of cell-cell contacts, expression of EMT markers, increased cell motility and increased resistance to doxorubicin or docetaxel. Experimental modulation of CD146 expression induces changes consistent with the above described characteristics: morphology, motility, growth in anchorage independent conditions and Slug mRNA variations are strictly correlated with CD146 expression. These changes are associated with modifications of ER (estrogen receptor) and Erb receptors and are enhanced by simultaneous and opposite modulation of JAM-A, or exposure to heregulin, an erb-B4 ligand.

CONCLUSIONS

CD146 expression is associated with an EMT phenotype. Several molecules are affected by CD146 expression: direct or indirect signaling contributes to EMT by increasing Slug expression. CD146 may also interact with Erb signaling by modifying cell surface expression of ErbB3 and ErbB4 and increased resistance to chemotherapy. Antagonistic effects of JAM-A, a tight junction-associated protein, on CD146 promigratory effects underline the complexity of the adhesion molecules network in tumor cell migration and metastasis.

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G(1) arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G(1) arrest. This G(1) arrest was associated with up-regulation of p27(kip1), whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G(1) arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 ΔEGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.