BACKGROUND

Epidermal growth factor receptor (EGFR) signaling pathway plays a crucial role in the development and progression of lung cancer. We searched for mutations of EGFR pathway genes in non-small cell lung cancers (NSCLCs) and analyzed their relationship with clinicopathologic features.

METHODS

Mutations of EGFR, ERBB2, ERBB3, ERBB4, KRAS, NRAS, BRAF, PTEN, PIK3CA, LKB1, and AKT1 genes were determined by direct sequencing in 173 surgically resected NSCLCs--56 squamous cell carcinomas (SCCs) and 117 adenocarcinomas (ACs).

RESULTS

Of the 173 NSCLCs, a total of 65 mutations were detected in 63 (36.4%) tumors--10 (17.9%) in SCCs and 53 (45.3%) in ACs. Mutations in EGFR pathway genes were significantly more frequent in women and ACs than in women and SCCs (p = 0.02 and p < 0.001, respectively). The mutations occurred in a mutually exclusive pattern. When the genes were divided into three subgroups according to their roles in the signaling cascade, mutations in the EGFR/ERBB2 and KRAS/BRAF genes were more frequent in ACs than in SCCs (p < 0.001 and p = 0.01, respectively). In marked contrast, mutations in the PIK3CA/PTEN were more frequent in SCCs than in ACs (p = 0.002). Furthermore, mutations in the PIK3CA/PTEN genes were more frequent in smokers (p = 0.04).

CONCLUSIONS

Our study demonstrates that mutations in each part of the EGFR pathway were associated with different clinicopathologic features in patients with NSCLCs.

The ErbB family, ErbB1 (also known as the epidermal growth factor receptor EGFR), ErbB2, ErbB3, and ErbB4 comprise a group of receptor tyrosine kinases that interact with ligands from the epidermal growth factor (EGF) superfamily, subsequently dimerize, catalytically activate each other by cross-phosphorylation, and then stimulate various signaling pathways. To gain a better understanding of in vivo functions of ErbB receptors in the central nervous system, the current study examined their mRNA expression throughout development in the mouse brain via in situ hybridization. EGFR, ErbB2, and ErbB4 exhibited distinct but sometimes overlapping distributions in multiple cell types within germinal zones, cortex, striatum, and hippocampus in prenatal and postnatal development. In addition, a subpopulation of cells positive for ErbB4 mRNA in postnatal cortex and striatum coexpressed mRNA for either EGFR or GAD67, a marker for gamma-aminobutyric acid (GABA)ergic interneurons, suggesting that both ErbB4 and EGFR are coexpressed in GABAergic interneurons. In contrast, ErbB3 mRNA was not detected within the brain during development and only appeared in white matter tracts in adulthood. Together, these findings suggest that ErbB receptors might mediate multiple functions in central nervous system development, some of which may be initiated by EGFR/ErbB4 heterodimers in vivo.

Patients with neurofibromatosis type 1 develop aggressive Schwann cell neoplasms known as malignant peripheral nerve sheath tumors (MPNSTs). Although tumor suppressor gene mutations play an important role in MPNST pathogenesis, it is likely that dysregulated signaling by as yet unidentified growth factors also contributes to the formation of these sarcomas. To test the hypothesis that neuregulin-1 (NRG-1) growth factors promote mitogenesis in MPNSTs, we examined the expression and action of NRG-1 in human MPNSTs and neurofibromas, the benign precursor lesions from which MPNSTs arise. Multiple alpha and beta transmembrane precursors from the class II and III NRG-1 subfamilies are present in both tumor types. Neoplastic Schwann cells within these neoplasms variably express the erbB kinases mediating NRG-1 responses (erbB2, erbB3 and/or erbB4). Human MPNST cell lines (Mash-1, YST-1, NMS-2 and NMS-2PC cells) similarly coexpress multiple NRG-1 isoforms and erbB receptors. These MPNST lines are NRG-1 responsive and demonstrate constitutive erbB phosphorylation. Treatment with PD168393 and PD158780, two structurally and mechanistically distinct erbB inhibitors, abolishes erbB phosphorylation and reduces DNA synthesis in these lines. These findings suggest that autocrine and/or paracrine NRG-1/erbB signaling promotes neoplastic Schwann cell proliferation and may be an important therapeutic target in neurofibromas and MPNSTs.

Childhood-onset schizophrenia (COS), defined as onset of psychosis by the age of 12, is a rare and malignant form of the illness, which may have more salient genetic influence. Since the initial report of association between neuregulin 1 (NRG1) and schizophrenia in 2002, numerous independent replications have been reported. In the current study, we genotyped 56 markers (54 single-nucleotide polymorphisms (SNPs) and two microsatellites) spanning the NRG1 locus on 78 COS patients and their parents. We used family-based association analysis for both diagnostic (extended transmission disequilibrium test) and quantitative phenotypes (quantitative transmission disequilibrium test) and mixed-model regression. Most subjects had prospective anatomic brain magnetic resonance imaging (MRI) scans at 2-year intervals. Further, we genotyped a sample of 165 healthy controls in the MRI study to examine genetic risk effects on normal brain development. Individual markers showed overtransmission of alleles to affecteds (P=0.009-0.05). Further, several novel four-marker haplotypes demonstrated significant transmission distortion. There was no evidence of epistasis with SNPs in erbB4. The risk allele (0) at 420M9-1395 was associated with poorer premorbid social functioning. Further, possession of the risk allele was associated with different trajectories of change in lobar volumes. In the COS group, risk allele carriers had greater total gray and white matter volume in childhood and a steeper rate of subsequent decline in volume into adolescence. By contrast, in healthy children, possession of the risk allele was associated with different trajectories in gray matter only and was confined to frontotemporal regions, reflecting epistatic or other illness-specific effects mediating NRG1 influence on brain development in COS. This replication further documents the role of NRG1 in the abnormal brain development in schizophrenia. This is the first demonstration of a disease-specific pattern of gene action in schizophrenia.

Heregulin (HRG) is a soluble secreted growth factor, which, upon binding and activation of ErbB3 and ErbB4 transmembrane receptor tyrosine kinases, is involved in cell proliferation, invasion, survival and differentiation of normal and malignant tissues. The HRG gene family consists of four members: HRG-1, HRG-2, HRG-3 and HRG-4, of which a multitude of different isoforms are synthesized by alternative exon splicing, showing various tissue distribution and biological activities. Disruption of the physiological balance between HRG ligands and their ErbB receptors is implicated in the formation of a variety of human cancers. The general mechanisms involved in HRG-induced tumorigenesis is discussed.

Alterations in the expression of the epidermal growth factor (EGF) receptor ErbB family are frequently encountered in a number of human cancers. Two of these receptors, ErbB3 and ErbB4, are known to bind a family of related proteins termed heregulins (HRGs) or neu differentiation factors. In biologically relevant systems, interaction of HRG with ErbB3 or ErbB4 results in the transactivation of ErbB2. In this report, we show that ErbB2 is a critical component in mediating HRG responsiveness in a panel of human breast and ovarian tumor cell lines. Because HRGs have been reported to elicit diverse biological effects on cultured cells, including growth stimulation, growth inhibition, and induction of differentiation, we systematically examined the effect of rHRG beta 1 on tumor cell proliferation. HRG binding studies were performed with a panel of breast and ovarian tumor cell lines expressing a range of levels of ErbB2. The biological responses to HRG were also compared to EGF and to the growth-inhibitory anti-ErbB2 antibody, 4D5. In most cases, HRG stimulation of DNA synthesis correlated with positive effects on cell cycle progression and cell number and with enhancement of colony formation in soft agar. On each cell line tested, the HRG effects were distinguishable from EGF and 4D5. Our findings indicate that HRG induces cell proliferation in a number of tumor cell lines. In addition, we show that methods for measuring cell proliferation, as well as experimental conditions, are critical for determining HRGs effect on tumor cell growth in vitro.

Membrane-bound and secreted neuregulin isoforms induce growth, survival and differentiation by activating erbB tyrosine kinase receptors. In cultured cardiomyocytes, erbB2 and erbB4 receptors regulate apoptosis by controlling bcl-x splicing, and conditional elimination of erbB2 induces dilative cardiomyopathy in vivo. Therefore, we analyzed expression and activation of erbB receptors in left ventricular myocardium from 32 heart failure patients, from 10 organ donors, and from 15 heart failure patients prior to and following unloading by ventricular assist devices. ErbB receptors, expressed in cardiomyocytes and noncardiomyocytes, are downregulated in failing myocardium as mRNA (which is renormalized by hemodynamic unloading) and as protein (erbB2: -25%; erbB4: -70%), their phosphorylation is reduced and bcl-x splicing is shifted towards 6.7-fold augmentation of proapoptotic Bcl-xS, compatible with attenuated erbB signaling. However, secreted and membrane-anchored neuregulin-1 isoforms, preferentially expressed in microvascular endothelium, are induced and not lowered with heart failure, while expression of erbB-inhibitory neuregulin isoforms or of autoinhibitory soluble erbB isoforms could not be demonstrated as potential causes of erbB receptor inhibition. We conclude that erbB receptor inactivation by unknown mechanisms results in altered splicing of bcl-x towards enhanced formation of proapoptotic Bcl-xS, thereby contributing to enhanced apoptotic susceptibility of failing human myocardium.

The p75 neurotrophin receptor (p75(NTR)), a member of the tumor necrosis factor superfamily of receptors, undergoes multiple proteolytic cleavage events. These events are initiated by an alpha-secretase-mediated release of the extracellular domain followed by a gamma-secretase-mediated intramembrane cleavage. However, the specific determinants of p75(NTR) cleavage events are unknown. Many other substrates of gamma-secretase cleavage have been identified, including Notch, amyloid precursor protein, and ErbB4, indicating there is broad substrate recognition by gamma-secretase. Using a series of deletion mutations and chimeric receptors of p75(NTR) and the related Fas receptor, we have identified domains that are essential for p75(NTR) proteolysis. The initial alpha-secretase cleavage was extracellular to the transmembrane domain. Unfortunately, deletion mutants were not capable of defining the requirements of ectodomain shedding. Although this cleavage is promiscuous with respect to amino acid sequence, its position with respect to the transmembrane domain is invariant. The generation of chimeric receptors exchanging different domains of noncleavable Fas receptor with p75(NTR), however, revealed that a discrete domain above the membrane is sufficient for efficient cleavage of p75(NTR). Mass spectrometric analysis confirmed the cleavage can occur with a truncated p75(NTR) displaying only 15 extracellular amino acids in the stalk region.

Neuregulin 1 (NRG1) is a secreted trophic factor that activates the postsynaptic erbB4 receptor tyrosine kinase. Both NRG1 and erbB4 have been repeatedly associated with schizophrenia, but their downstream targets are not well characterized. ErbB4 is highly abundant in interneurons, and NRG1-mediated erbB4 activation has been shown to modulate interneuron function, but the role for NRG1-erbB4 signaling in regulating interneuron dendritic growth is not well understood. Here we show that NRG1/erbB4 promote the growth of dendrites in mature interneurons through kalirin, a major dendritic Rac1-GEF. Recent studies have shown associations of the KALRN gene with schizophrenia. Our data point to an essential role of phosphorylation in kalirin-7's C terminus as the critical site for these effects. As reduced interneuron dendrite length occurs in schizophrenia, understanding how NRG1-erbB4 signaling modulates interneuron dendritic morphogenesis might shed light on disease-related alterations in cortical circuits.

ErbB-4 is a recently discovered member of the class I receptor tyrosine kinase family (ErbB). Little is known about its expression and its importance in human malignancy. To delineate the biological function of ErbB-4 receptors in breast cancer, we used a hammerhead ribozyme strategy to achieve down-regulation of ErbB-4 receptors in various breast cancer cell lines. We observed that down-regulation of ErbB-4 in estrogen receptor-positive (ER+) human breast cancer cell lines (MCF-7 and T47D), which express relatively high levels of ErbB-4, significantly inhibited colony formation. No effects were observed in estrogen receptor-negative (ER-) MDA-MB-453 cells, which express low levels of endogenous ErbB4 and high levels of ErbB-2 and ErbB-3. This occurred despite the fact that fluorescence-activated cell sorter analysis of these latter cells revealed that the expression of the ErbB-4 receptor was completely abrogated by ribozyme treatment. Furthermore, down-regulation of ErbB-4 in T47D and MCF-7 cells significantly inhibited tumor formation in athymic nude mice (P < 0.03 and P < 0.001, respectively). In addition, NRG-stimulated phosphorylation of ErbB-4- and NRG-induced colony formation was significantly reduced in ribozyme-transfected T47D cells. These data provide the first evidence that elevation of ErbB-4 expression plays a role in the proliferation of some ER+ human breast cancer cell lines (T47D and MCF-7) that express high levels of ErbB-4. We have also investigated the expression of ErbB-4 in human primary breast carcinoma specimens, using immunohistochemical staining with an anti-ErbB-4 monoclonal antibody. ErbB-4 expression was found in 60% of the 50 primary breast tumors examined, and high intense immunoreactivity of ErbB-4 was detected in 18% of these primary breast tumors. ErbB-4 receptor expression appeared to correlate with ER+ primary breast tumors. A similar correlation was also observed in the human breast cancer cell lines. These results provide a better understanding of the biological significance of ErbB-4 receptor in breast cancer. Our data suggest that elevation of the ErbB-4 receptor plays a role in ER+ breast cancer cell proliferation. Moreover, ribozyme technology provides a useful tool to delineate the role of a particular gene product.

Prepulse inhibition (PPI) of the startle response is an important measure of information processing deficits and inhibitory failure in schizophrenia patients. PPI is especially useful because it occurs in the same lawful manner in all mammals, from humans to rodents, making it an ideal candidate for cross-species translational research. PPI deficits occur across the "schizophrenia spectrum" from schizophrenia patients to their clinically unaffected relatives. Parallel animal model and human brain imaging studies have demonstrated that PPI is modulated by cortico-striato-pallido-thalamic (and pontine) circuitry. This circuitry is also implicated in schizophrenia neuropathology and neurophysiology. The finding of PPI deficits in schizophrenia patients has been replicated by many groups, and these deficits correlate with measures of thought disorder and appear to be "normalized" by second generation antipsychotic (SGA) medications. Consistent pharmacological effects on PPI have been demonstrated; among these, dopamine agonists induce PPI deficits and (in animal models) these are reversed by first and SGA medications. PPI is also significantly heritable in humans and animals and can be used as a powerful endophenotype in studies of families of schizophrenia patients. Genomic regions, including the NRGL-ERBB4 complex with its glutamatergic influences, are strongly implicated in PPI deficits in schizophrenia. PPI continues to hold promise as an exciting translational cross-species measure that can be used to understand the pathophysiology and treatment of the schizophrenias via pharmacological, anatomic, and genetic studies.

Heregulins are a family of ligands for the ErbB3/ErbB4 receptors that play important roles in breast cancer cell proliferation and tumorigenesis. Limited information is available on the contribution of Rho GTPases to heregulin-mediated signaling. In breast cancer cells, heregulin beta1 (HRG) causes a strong activation of Rac; however, it does so with striking differences in kinetics compared to epidermal growth factor, which signals through ErbB1 (epidermal growth factor receptor [EGFR]). Using specific ErbB receptor inhibitors and depletion of receptors by RNA interference (RNAi), we established that, surprisingly, activation of Rac by HRG is mediated not only by ErbB3 and ErbB2 but also by transactivation of EGFR, and it is independent of ErbB4. Similar receptor requirements are observed for HRG-induced actin cytoskeleton reorganization and mitogenic activity via extracellular signal-regulated kinase (ERK). HRG-induced Rac activation was phosphatidylinositol 3-kinase dependent and Src independent. Furthermore, inactivation of Rac by expression of the Rac GTPase-activating protein beta2-chimerin inhibited HRG-induced ERK activation, mitogenicity, and migration in breast cancer cells. HRG mitogenic activity was also impaired by depletion of Rac1 using RNAi. Our studies established that Rac is a critical mediator of HRG mitogenic signaling in breast cancer cells and highlight additional levels of complexity for ErbB receptor coupling to downstream effectors that control aberrant proliferation and transformation.

Neuregulin and the neuregulin receptor ERBB4 have been genetically and functionally implicated in schizophrenia. In this study, we used the yeast two-hybrid system to identify proteins that interact with ERBB4, to identify genes and pathways that might contribute to schizophrenia susceptibility. We identified the MAGI scaffolding proteins as ERBB4-binding proteins. After validating the interaction of MAGI proteins with ERBB4 in mammalian cells, we demonstrated that ERBB4 expression, alone or in combination with ERBB2 or ERBB3, led to the tyrosine phosphorylation of MAGI proteins, and that this could be further enhanced with receptor activation by neuregulin. As MAGI proteins were previously shown to interact with receptor phosphotyrosine phosphatase beta/zeta (RPTPbeta), we postulated that simultaneous binding of MAGI proteins to RPTPbeta and ERBB4 forms a phosphotyrosine kinase/phosphotyrosine phosphatase complex. Studies in cultured cells confirmed both a spatial and functional association between ERBB4, MAGI and RPTPbeta. Given the evidence for this functional association, we examined the genes coding for MAGI and RPTPbeta for genetic association with schizophrenia in a Caucasian United Kingdom case-control cohort (n= approximately 1400). PTPRZ1, which codes for RPTPbeta, showed significant, gene-wide and hypothesis-wide association with schizophrenia in our study (best individual single-nucleotide polymorphism allelic P=0.0003; gene-wide P=0.0064; hypothesis-wide P=0.026). The data provide evidence for a role of PTPRZ1, and for RPTPbeta signaling abnormalities, in the etiology of schizophrenia. Furthermore, the data indicate a role for RPTPbeta in the modulation of ERBB4 signaling that may in turn provide further support for an important role of neuregulin/ERBB4 signaling in the molecular basis of schizophrenia.

The signaling complex consisting of the growth factor neuregulin-1 (NRG1) and its tyrosine kinase receptors ErbB2 and ErbB4 has a critical role in cardiac development and homeostasis of the structure and function of the adult heart. Recent research results suggest that targeting this signaling complex may provide a viable strategy for treating heart failure. Clinical trials are currently evaluating the effectiveness and safety of intravenous administration of recombinant NRG1 formulations in heart failure patients. Endogenous as well as administered NRG1 has multiple possible activities in the adult heart, but how these are related is unknown. It has recently been demonstrated that NRG1 administration can stimulate proliferation of cardiomyocytes, which may contribute to repair failing hearts. This review summarizes the current knowledge of how NRG1 and its receptors control cardiac physiology and biology, with special emphasis on its role in cardiomyocyte proliferation during myocardial growth and regeneration.

Neuregulin-1 (NRG1) is implicated in the etiology or pathology of schizophrenia, although its biological roles in this illness are not fully understood. Human midbrain dopaminergic neurons highly express NRG1 receptors (ErbB4). To test its neuropathological role in the neurodevelopmental hypothesis of schizophrenia, we administered type-1 NRG1 protein to neonatal mice and evaluated the immediate and subsequent effects on dopaminergic neurons and their associated behaviors. Peripheral NRG1 administration activated midbrain ErbB4 and elevated the expression, phosphorylation and enzyme activity of tyrosine hydroxylase (TH), which ultimately increased dopamine levels. The hyperdopaminergic state was sustained in the medial prefrontal cortex after puberty. There were marked increases in dopaminergic terminals and TH levels. In agreement, higher amounts of dopamine were released from this brain region of NRG1-treated mice following high potassium stimulation. Furthermore, NRG1-treated mice exhibited behavioral impairments in prepulse inhibition, latent inhibition, social behaviors and hypersensitivity to methamphetamine. However, there were no gross abnormalities in brain structures or other phenotypic features of neurons and glial cells. Collectively, our findings provide novel insights into neurotrophic contribution of NRG1 to dopaminergic maldevelopment and schizophrenia pathogenesis.

Putative roles of neuregulin (NRG) and the ErbB receptors in skeletal muscle biology include myogenesis, ACh receptor expression, and glucose transport. To date, however, the physiological regulation of NRG/ErbB signaling has not been examined. We tested the hypothesis that contractile activity in vivo induces NRG/ErbB activation. Rat hindlimb muscle contraction was elicited with a single bout of electrical stimulation (RX) or treadmill running (EX). Western blot and immunofluorescence confirmed the expression of multiple NRG isoforms and the ErbB2, ErbB3, and ErbB4 receptors in adult skeletal muscle. Both RX and EX significantly increased phosphorylation of all NRG receptors. Furthermore, contraction induced a shift in the expression profile of NRG, consistent with proteolytic processing of a transmembrane isoform. Thus two distinct modes of exercise activated processing of NRG with concomitant stimulation of ErbB2, ErbB3, and ErbB4 signaling in vivo. To our knowledge, this is the first demonstration of physiological regulation of NRG/ErbB signaling in any organ and implicates this pathway in the metabolic and proliferative responses of skeletal muscle to exercise.

Tumor necrosis factor-alpha converting enzyme (TACE) is a membrane-anchored, Zn-dependent metalloprotease, which belongs to the ADAM (a disintegrin and metalloprotease) family. TACE functions as a membrane sheddase to release the ectodomain portions of many transmembrane proteins, including the precursors of TNFalpha, TGFalpha, several other cytokines, as well as the receptors for TNFalpha, and neuregulin (ErbB4). Mice with TACE(DeltaZn/DeltaZn) null mutation die at birth with phenotypic changes, including failure of eyelid fusion, hair and skin defects, and abnormalities of lung development. Abnormal fetal heart development was not previously described. Herein, we report that TACE(DeltaZn/DeltaZn) null mutant mice by late gestation exhibit markedly enlarged fetal hearts with increased myocardial trabeculation and reduced cell compaction, mimicking the pathological changes of noncompaction of ventricular myocardium. In addition, larger cardiomyocyte cell size and increased cell proliferation were observed in ventricles of TACE(DeltaZn/DeltaZn) knockout mouse hearts. At the molecular level, reduced expression of epidermal growth factor receptor, attenuated protein cleavage of ErbB4, and changes in MAPK activation were also detected in TACE(DeltaZn/DeltaZn) knockout heart tissues. The data suggest that TACE-mediated cell surface protein ectodomain shedding plays an essential and a novel regulatory role during cardiac development and modeling.

The formation of the neuromuscular junction (NMJ) is directed by reciprocal interactions between motor neurons and muscle fibers. Neuregulin (NRG) and Agrin from motor nerve terminals are both implicated. Here, we demonstrate that NMJs can form in the absence of the NRG receptors ErbB2 and ErbB4 in mouse muscle. Postsynaptic differentiation is, however, induced by Agrin. We therefore conclude that NRG signaling to muscle is not required for NMJ formation. The effects of NRG signaling to muscle may be mediated indirectly through Schwann cells.

Epidermal growth factor receptor (EGFR), erbB2, erbB3 and erbB4 are four transmembrane glycoproteins belonging to the subtype I tyrosine kinases. They share structure homologies and are believed to direct cellular growth through the ligand-stimulated tyrosine phosphorylation of intracellular substrate. The overexpression of these tyrosine kinases has been linked to various cancers. To examine the role of the erbB family in the neoplastic transformation of the human colon, we analysed the protein expression of these four members by immunohistochemistry in paraffin-embedded specimens from 125 resected colorectal cancers. Our data showed that for EGFR expression, 62 (50%) were scored as '+', and 2 (2%) as '++'. For erbB2 expression, 39 (31%) were classified as '+', and 5 (4%) as '++'. For erbB3 expression, 43 (34%) were scored as '+', and 3 (2%) as '++'. A significantly higher percentage of overexpressed erbB3 was observed in early stage carcinomas (Dukes' stage A or B) (50%) than in advanced stage cancers (Dukes' stage C or D) (15%) (P<0.0001). For erbB4 expression, 22 (18%) were scored as '+', and 5 (4%) as '++'. Early stage patients had a lower percentage of erbB4 overexpression than the late stage ones (18% versus 28%). Concomitant overexpression of erbB2 and erbB3 occurred in 21% (16/78) of the early stage carcinomas, whereas it occurred in only 2% (1/47) of the late stage ones (P=0.003). Conversely, simultaneous overexpression of erbB2 and erbB4 occurred in 17% (8/47) of the late stage carcinomas but in only 4% (3/78) of the early stage ones (P=0.02). Overexpression of EGFR, erbB2, erbB3 or erbB4 alone was not significantly associated with a shortened survival. However, patients with a simultaneous overexpression of erbB2 and erbB4 had a shorter overall survival time than others in the univariate analysis (P=0.01). This significance disappeared after adjustment for Dukes' staging in the Cox model. In conclusion, overexpressed erbB3 was common in early stage colorectal cancers, but its prevalence was significantly reduced in late stage ones. The percentage of its coexpression with erbB2 was significantly higher in early stage than in late-stage cancers. Heterodimerisation between erbB2 and erbB4 may play a role in the late stages of carcinogenesis.

The epidermal growth factor receptor (EGFR, c-erbB1) plays a pivotal role in maintenance and repair of epithelial tissues; however, little is known about coexpression of c-erbB receptors and their ligands in human bronchial epithelium. We therefore analyzed the expression of these molecules in cultured bronchial epithelial cells and normal bronchial mucosa, using reverse transcription-polymerase chain reaction (RT- PCR), flow cytometry, and immunohistochemistry. Messenger RNA (mRNA) encoding EGFR, c-erbB2, and c-erbB3, but not c-erbB4, was detected in primary cultures of human bronchial epithelial cells, as well as in the human bronchial epithelial-derived cell lines H292 and 16HBE 14o-. Transcripts encoding epidermal growth factor (EGF), heparin binding epidermal growth factor (HB-EGF), transforming growth factor-alpha (TGF-alpha), and amphiregulin (AR) were also detected, and expression of the three receptors and four ligands was confirmed by immunocytochemical staining of the cultured cells. Immunohistochemical analysis of resin- or paraffin-embedded sections from surgical specimens of bronchial mucosa revealed strong membrane staining for EGFR within the bronchial epithelium; this was particularly evident between basal cells and the basal aspect of columnar cells. The patterns of staining for c-erbB2 and c-erbB3 in the bronchial epithelium were similar to those for EGFR. Immunostaining for EGF, TGF-alpha, AR, HB- EGF, and betacellulin (BTC) was intense in the submucosal glands; with the exception of BTC, EGFR ligand immunoreactivity was also observed in the bronchial epithelium, where it paralleled EGFR staining. Colocalization of c-erbB receptors and ligands demonstrates the potential for productive c-erbB receptor interactions in bronchial epithelium. Further study of these interactions may help to define their role in maintenance and repair of the bronchial epithelium.

The significant impact of microRNAs (miRNAs) on disease pathology is becoming increasingly evident. These small non-coding RNAs have the ability to post-transcriptionally silence the expression of thousands of genes. Therefore, dysregulation of even a single miRNA could confer a large polygenic effect. Schizophrenia is a genetically complex illness thought to involve multiple genes each contributing a small risk. Large genome-wide association studies identified miR-137, a miRNA shown to be involved in neuronal maturation, as one of the top risk genes. To assess the potential mechanism of impact of miR-137 in this disorder and identify its targets, we used a combination of literature searches, ingenuity pathway analysis (IPA), and freely accessible bioinformatics resources. Using TargetScan and the schizophrenia gene resource (SZGR) database, we found that in addition to CSMD1, C10orf26, CACNA1C, TCF4, and ZNF804A, five schizophrenia risk genes whose transcripts are also validated miR-137 targets, there are other schizophrenia-associated genes that may be targets of miR-137, including ERBB4, GABRA1, GRIN2A, GRM5, GSK3B, NRG2, and HTR2C. IPA analyses of all the potential targets identified several nervous system (NS) functions as the top canonical pathways including synaptic long-term potentiation, a process implicated in learning and memory mechanisms and recently shown to be altered in patients with schizophrenia. Among the subset of targets involved in NS development and function, the top scoring pathways were ephrin receptor signaling and axonal guidance, processes that are critical for proper circuitry formation and were shown to be disrupted in schizophrenia. These results suggest that miR-137 may indeed play a substantial role in the genetic etiology of schizophrenia by regulating networks involved in neural development and brain function.

ERBB4, one of four ErbB receptor tyrosine kinase family members, plays an important role in the etiology and progression of lung cancer. In this study, we found that the ERBB4 protein levels were consistently up-regulated in lung cancer tissues, whereas the mRNA levels varied randomly, suggesting that a post-transcriptional mechanism was involved in regulating ERBB4 expression. Because microRNAs are powerful post-transcriptional regulators of gene expression, we used bioinformatic analyses to search for microRNAs that can potentially target ERBB4. We identified specific targeting sites for miR-193a-3p in the 3'-UTR of ERBB4. We further identified an inverse correlation between miR-193a-3p levels and ERBB4 protein levels, but not mRNA levels, in lung cancer tissue samples. By overexpressing or knocking down miR-193a-3p in lung cancer cells, we experimentally confirmed that miR-193a-3p directly recognizes the 3'-UTR of the ERBB4 transcript and regulates ERBB4 expression. Furthermore, the biological consequences of the targeting of ERBB4 by miR-193a-3p were examined in vitro via cell proliferation, invasion, and apoptosis assays and in vivo using a mouse xenograft tumor model. We demonstrated that the repression of ERBB4 by miR-193a-3p suppressed proliferation and invasion and promoted apoptosis in lung cancer cells and that miR-193a-3p exerted an anti-tumor effect by negatively regulating ERBB4 in xenograft mice. Taken together, our findings provide the first clues regarding the role of miR-193a-3p as a tumor suppressor in lung cancer through the inhibition of ERBB4 translation.

Glioma is the most common type of adult brain tumor and glioblastoma, its most aggressive form, has a dismal prognosis. Receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR, ERBB2, ERBB3, ERBB4) family, and the vascular endothelial growth factor receptor (VEGFR), play a central role in tumor progression. We investigated the genetic variants of EGFR, ERBB2, VEGFR and their ligands, EGF and VEGF on glioma and glioblastoma risk. In addition, we evaluated the association of genetic variants of a newly discovered family of genes known to interact with EGFR: LRIG2 and LRIG3 with glioma and glioblastoma risk. Methods. We analyzed 191 tag single nucleotide polymorphisms (SNPs) capturing all common genetic variation of EGF, EGFR, ERBB2, LRIG2, LRIG3, VEGF and VEGFR2 genes. Material from four case-control studies with 725 glioma patients (329 of who were glioblastoma patients) and their 1 610 controls was used. Haplotype analyses were conducted using SAS/Genetics software. Results. Fourteen of the SNPs were significantly associated with glioma risk at p< 0.05, and 17 of the SNPs were significantly associated with glioblastoma risk at p< 0.05. In addition, we found that one EGFR haplotype was related to increased glioblastoma risk at p=0.009, Odds Ratio [OR] = 1.67 (95% confidence interval (CI): 1.14, 2.45). The Bonferroni correction made all p-values non-significant. One SNP, rs4947986 next to the intron/exon boundary of exon 7 in EGFR, was validated in an independent data set of 713 glioblastoma and 2 236 controls, [OR] = 1.42 (95% CI: 1.06,1.91). Discussion. Previous studies show that regulation of the EGFR pathway plays a role in glioma progression but the present study is the first to find that certain genotypes of the EGFR gene may be related to glioblastoma risk. Further studies are required to reinvestigate these findings and evaluate the functional significance.

Stochastic processes and imprinting, along with genetic factors, lead to monoallelic or allele-biased gene expression. Stochastic monoallelic expression fine-tunes information processing in immune cells and the olfactory system, and imprinting plays an important role in development. Recent studies suggest that both stochastic events and imprinting may be more widespread than previously considered. We are interested in allele-biased gene expression occurring in the brain because parent-of-origin effects suggestive of imprinting appear to play a role in the transmission of schizophrenia (SZ) and autism spectrum disorders (ASD) in some families. In addition, allele-biased expression could help explain monozygotic (MZ) twin discordance and reduced penetrance. The ability to study allele-biased expression in human neurons has been transformed with the advent of induced pluripotent stem cell (iPSC) technology and next generation sequencing. Using transcriptome sequencing (RNA-Seq) we identified 801 genes in differentiating neurons that were expressed in an allele-biased manner. These included a number of putative SZ and ASD candidates, such as A2BP1 (RBFOX1), ERBB4, NLGN4X, NRG1, NRG3, NRXN1, and NLGN1. Overall, there was a modest enrichment for SZ and ASD candidate genes among those that showed evidence for allele-biased expression (chi-square, p = 0.02). In addition to helping explain MZ twin discordance and reduced penetrance, the capacity to group many candidate genes affecting a variety of molecular and cellular pathways under a common regulatory process - allele-biased expression - could have therapeutic implications.