Overactivation of the ErbB protein family, which is comprised of 4 receptor tyrosine kinase members (ErbB1/epidermal growth factor receptor [EGFR]/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), can drive the development and progression of a wide variety of malignancies, including colorectal, head and neck, and certain non-small cell lung cancers (NSCLCs). As a result, agents that target a specific member of the ErbB family have been developed for the treatment of cancer. These agents include the reversible EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib; the EGFR-targeting monoclonal antibodies cetuximab and panitumumab; and the HER2-targeting monoclonal antibody trastuzumab. Lapatinib is a dual TKI that targets both EGFR and HER2. In addition, TKIs that inhibit multiple members of the ErbB family and also bind their targets irreversibly are under evaluation for the treatment of cancer. Three such compounds have progressed into clinical studies: the EGFR, HER2, and HER4 inhibitors afatinib, dacomitinib, and neratinib. Phase I studies of these agents have shown clinical activity in NSCLC, breast cancer, and other malignancies. Currently, afatinib is approved for EGFR mutation-positive NSCLC and is in development for squamous NSCLC, and dacomitinib is in phase III of clinical development for NSCLC, neratinib is in phase III of clinical development for the treatment of breast cancer, and afatinib is also in phase III development in head and neck cancer. Final results from clinical trials may lead to the potential approval of these agents in a variety of solid tumor malignancies.

Neuregulin-1 (NRG1) participates in numerous neurodevelopmental processes and plasticity of the brain. Despite this, little is known about its role in Alzheimer's disease (AD). Amyloid β (Aβ) peptide is generally believed to play a critical role in the pathogenesis of AD. The present study examined the effect of synthetic Aβ₁₋₄₂ peptides on long-term potentiation (LTP) in the CA1 region of mice hippocampal slices, a cellular model of learning and memory. We found that application of a test dose of Aβ₁₋₄₂ (200 nM) significantly inhibited the development of LTP without affecting basal synaptic transmission. Pretreatment with NRG1 effectively prevented Aβ₁₋₄₂-induced impairment of LTP, an effect that was dose-dependent. This LTP-restoring action of NRG1 was almost completely abolished by blocking ErbB4, a key NRG1 receptor, suggesting that NRG1 acts through ErbB4 to exert its protective action on LTP. The present study thus provides the first demonstration that NRG1/ErbB4 protects against Aβ-induced hippocampal LTP impairment, suggesting that NRG1 may be a promising candidate for the treatment of early-stage AD.

Growth factor Neuregulin 1 (NRG1) plays an essential role in development and organization of the cerebral cortex. NRG1 and its receptors, ERBB3 and ERBB4, have been implicated in genetic susceptibility for schizophrenia. Disease symptoms include asociality and altered social interaction. To investigate the role of NRG1-ERBB signaling in social behavior, mice heterozygous for an Nrg1 null allele (Nrg1+/-), and mice with conditional ablation of Erbb3 or Erbb4 in the central nervous system, were evaluated for sociability and social novelty preference in a three-chambered choice task. Results showed that deficiencies in NRG1 or ERBB3 significantly enhanced sociability. All of the mutant groups demonstrated a lack of social novelty preference, in contrast to their respective wild-type controls. Effects of NRG1, ERBB3, or ERBB4 deficiency on social behavior could not be attributed to general changes in anxiety-like behavior, activity, or loss of olfactory ability. Nrg1+/- pups did not exhibit changes in isolation-induced ultrasonic vocalizations, a measure of emotional reactivity. Overall, these findings provide evidence that social behavior is mediated by NRG1-ERBB signaling.

Signaling by a variety of ligands including epidermal growth factor, betacellulin and neuregulin is mediated by the ErbB family of receptor tyrosine kinases. Studies on the prostate have shown that ErbB2 phosphorylation and signaling can be regulated by prostatic acid phosphatase, a histidine acid phosphatase which can dephosphorylate phospho-tyrosine residues in the ErbB2 receptor. Here we report that the histidine acid phosphatase ACPT (testicular acid phosphatase), which is highly homologous to the prostatic acid phosphatase, can dephosphorylate the ErbB4 receptor, which is known to play important roles in neuronal differentiation and synaptogenesis. ACPT and ErbB4 are both expressed in the brain where they are enriched at post-synaptic sites, and furthermore they can be co-immunoprecipitated from brain. We demonstrate that ACPT can inhibit basal and neuregulin-induced tyrosine phosphorylation of ErbB4. We also show that ACPT-dependent dephosphorylation can regulate the proteolytic cleavage of ErbB4, and this process can be reversed by applying the tyrosine phosphatase inhibitor, pervanadate. Furthermore, neuregulin-dependent differentiation of PC12 cells expressing ErbB4 is prevented by co-expressing ACPT. These results indicate that ACPT acts as a tyrosine phosphatase to modulate signals mediated by ErbB4 that are important for neuronal development and synaptic plasticity.

Epiregulin is an epidermal growth factor (EGF) member that activates ErBB1 and ErBB4 homodimers and all possible heterodimeric ErbB complexes. Because its role in renal cell regeneration has not been investigated, we assessed the effect of exogenous epiregulin on regeneration of renal proximal tubular cells (RPTC) in primary culture. Epiregulin (10 ng/ml) was equivalent to EGF (10 ng/ml) in enhancing RPTC proliferation and migration. Epiregulin induced activation of the EGF receptor (EGFR), Akt, a downstream kinase of phosphoinositide 3-kinase (PI3K), and extracellular signaling-regulated kinase 1/2 (ERK1/2). Treatment with AG1478, a specific EGFR inhibitor, blocked phosphorylation of EGFR, Akt, ERK1/2, proliferation, and migration. Furthermore, inactivation of PI3K with LY-294002 blocked epiregulin-induced RPTC proliferation and, to a lesser extent, migration. However, blockade of ERK1/2 had no such effects. We suggest that epiregulin is a potent mitogen for renal epithelial cells and may contribute to renal regeneration through activation of EGFR and PI3/Akt pathways.

As rapidly developing patient-derived xenografts (PDX) could represent potential sources of cancer stem cells (CSC), we selected and characterized non-cultured PDX cell suspensions from four different renal carcinomas (RCC). Only the cell suspensions from the serial xenografts (PDX-1 and PDX-2) of an undifferentiated RCC (RCC-41) adapted to the selective CSC medium. The cell suspension derived from the original tumor specimen (RCC-41-P-0) did not adapt to the selective medium and strongly expressed CSC-like markers (CD133 and CD105) together with the non-CSC tumor marker E-cadherin. In comparison, PDX-1 and PDX-2 cells exhibited evolution in their phenotype since PDX-1 cells were CD133high/CD105-/Ecadlow and PDX-2 cells were CD133low/CD105-/Ecad-. Both PDX subsets expressed additional stem cell markers (CD146/CD29/OCT4/NANOG/Nestin) but still contained non-CSC tumor cells. Therefore, using different cell sorting strategies, we characterized 3 different putative CSC subsets (RCC-41-PDX-1/CD132+, RCC-41-PDX-2/CD133-/EpCAMlow and RCC-41-PDX-2/CD133+/EpCAMbright). In addition, transcriptomic analysis showed that RCC-41-PDX-2/CD133- over-expressed the pluripotency gene ERBB4, while RCC-41-PDX-2/CD133+ over-expressed several tumor suppressor genes. These three CSC subsets displayed ALDH activity, formed serial spheroids and developed serial tumors in SCID mice, although RCC-41-PDX-1/CD132+ and RCC-41-PDX-2/CD133+ displayed less efficiently the above CSC properties. RCC-41-PDX-1/CD132+ tumors showed vessels of human origin with CSC displaying peri-vascular distribution. By contrast, RCC-41-PDX-2 originated tumors exhibiting only vessels of mouse origin without CSC peri-vascular distribution.Altogether, our results indicate that PDX murine microenvironment promotes a continuous redesign of CSC phenotype, unmasking CSC subsets potentially present in a single RCC or generating ex novo different CSC-like subsets.

Similar to other epithelial appendages, mammary anlagen progress from stratified epithelium through placode and bud stages. Embryonic mammary morphogenesis is elicited by a combination of local cell migration, adhesion changes and proliferation, and these same developmental processes impact breast cancer etiology. The Erbb signaling network plays important roles in postnatal mammary gland morphogenesis and carcinogenesis. Neuregulin3 (Nrg3), an Erbb family ligand, has recently been shown to be involved in the specification of mammary glands in mice. To further examine the possible involvement of other Erbb family members and their ligands in early mammary morphogenesis, we have characterized their expression patterns during this process. We used whole mount in situ hybridization to analyze the expression patterns of these genes at stages prior to and during mammary placode formation. Immunohistochemistry was used to examine expression patterns at later bud stages. The Neuregulin ligands, Nrg1, Nrg2, Nrg3, Nrg4 and the receptors, Erbb1, Erbb2, Erbb3, Erbb4, were expressed either at stages prior to morphological appearance of the mammary placode or from the time that the placode is first morphologically distinct through to later bud stages. The expression patterns presented here suggest that multiple members of this signaling network are potential mediators of early mammary morphogenesis.

The rat olfactory bulb is an exceptional CNS tissue. Unlike other areas of the brain, growing axons are able to enter the olfactory bulb and extend within this CNS environment throughout adult life. It appears that the glial cells of the olfactory system, known as olfactory bulb ensheathing cells (OBECs), may have an important role in this remarkable process of CNS neural regeneration. OBECs are unusual glial cells, possessing properties of both astrocytes and Schwann cells. In this study we show that astrocytes (in the form of astrocyte-conditioned medium; ACM) produce two critical regulatory functions for OBECs: mitogenic activity and a survival factor. Interestingly, the ACM-derived activity for OBECs appears to reside in a signalling protein(s) belonging to the neuregulin (NRG) family of growth factors, and specifically appears to coincide with one or more products of the nrg-1 gene. Our observations provide evidence for the following: recombinant human neu differentiation factors (NDFbeta1, -2 and -3) are mitogenic to OBECs; the activity in ACM can be neutralized by NDF antibodies; these same antibodies detect a 50-kDa, non-heparin binding protein in concentrated ACM; astrocytes express detectable nrg-1 transcripts; and OBECs express functional NRG receptors erbB2 and erbB4.

Glioblastomas are the most common primary central nervous system tumors in adults, and they remain resistant to current treatments. erbB1 signaling is frequently altered in glioblastomas, suggesting thaterbB receptor family members may represent targets for molecular therapy. We performed a comprehensive analysis of erbB receptor and ligand expression profiles in a panel of 9 glioblastomas andcompared them to nonneoplastic cerebral tissue containing neocortex and adjacent white matter. Quantitative reverse transcription-polymerase chain reaction and Western blot analysis showed that erbB1signaling and erbB2 receptors exhibited highly variable deregulation profiles in the tumors, with patterns ranging from underexpression to overexpression; in contrast, erbB3 and erbB4 were downregulated. We next performed immunohistochemistry to determinethe distribution patterns of erbB receptors among the main neuralcell types in the tumors with special reference to the putative tumor stem cell population. Results revealed intertumoral and intratumoral heterogeneity in all 4 erbB expression profiles, but each receptor exhibited a distinct distribution pattern among glial fibrillary acidic protein-, Olig2-, NeuN-, and CD133-positive populations. Although erbB1 immunoreactivity was detected in only small subsets of CD133-positive putative tumor stem cells, erbB3 immunoreactivity was prominent in this population, suggesting that erbB3 may represent a new potential therapeutic target.

The neuregulin 1 (NRG1) receptor ErbB4 is involved in the development of cortical inhibitory GABAergic circuits and NRG1-ErbB4 signaling has been implicated in schizophrenia (SCZ). A magnetic resonance spectroscopy ((1)H-MRS) study has demonstrated that a single-nucleotide polymorphism in ERBB4, rs7598440, influences human cortical GABA concentrations. Other work has highlighted the significant impact of this genetic variant on expression of ERBB4 in the hippocampus and dorsolateral prefrontal cortex in human post mortem tissue. Our aim was to examine the association of rs7598440 with cerebrospinal fluid (CSF) GABA levels in healthy volunteers (n=155). We detected a significant dose-dependent association of the rs7598440 genotype with CSF GABA levels (G-allele standardized β=-0.23; 95% CIs: -0.39 to -0.07; P=0.0066). GABA concentrations were highest in A homozygous, intermediate in heterozygous, and lowest in G homozygous subjects. When excluding subjects on psychotropic medication (three subjects using antidepressants), the results did not change (G-allele standardized β=-0.23; 95% CIs: -0.40 to -0.07; P=0.0051). The explained variance in CSF GABA by rs7598440 in our model is 5.2% (P=0.004). The directionality of our findings agrees with the aforementioned (1)H-MRS and gene expression studies. Our observation therefore strengthens the evidence that the A-allele of rs7598440 in ERBB4 is associated with increased GABA concentrations in the human central nervous system (CNS). To our knowledge, our finding constitutes the first confirmation that CSF can be used to study genotype-phenotype correlations of GABA levels in the CNS. Such quantitative genetic analyses may be extrapolated to other CSF constituents relevant to SCZ in future studies.

The growth factor neuregulin 1 (NRG) selectively induces an increase in the gamma-aminobutyric acid (GABA)(A) receptor beta2 subunit protein in rat cerebellar granule neurons in culture. We previously demonstrated that NRG acts by triggering ErbB4 receptor phosphorylation and subsequent signaling through the mitogen-activated kinase (MAPK), phosphatidyl inositol-3 kinase (PI-3K) and cyclin-dependent kinase 5 (cdk5) pathways. In this report we show that the scaffolding protein, PSD-95, plays a key role in mediating the effects of NRG and that reducing its level attenuates the NRG-induced increase in beta2 subunit expression. PSD-95 appears to facilitate the effects of NRG through its association with ErbB4, an interaction that is augmented by NRG-activated cdk signaling. Inhibition of cdk activity with roscovitine attenuates the association of PSD-95 with ErbB4. The effects of cdk5 are not blocked by U0126, an inhibitor of MAPK signaling, indicating that cdk5 functions independently of cross-talk with this pathway. These findings raise the possibility that NRG-induced activation of cdk5 works in part by recruiting PSD-95, a protein involved in regulating synaptic plasticity, to associate with ErbB4. This interaction may be a positive feedback loop that augments NRG signaling and its downstream effects on GABA(A) receptor beta2 subunit expression.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant central nervous system neoplasm that usually affects infants and young children. In this report, we describe culture conditions that enabled the sustained growth of tumor cells obtained from the cerebrospinal fluid (CSF) of an infant with AT/RT. These cells retained the morphological and biomarker characteristics of the original tumor. A screening of receptor tyrosine kinases identified the presence of phosphorylated ErbB4, Insulin-R, PDGFR and IGF-IR, which appear to depend on Hsp90 to maintain their active form. IGF-IR activity is consistent with data from other established AT/RT cell lines. Inhibition of IGF-IR by the small molecular weight inhibitor AEW541 led to growth suppression of cultured AT/RT cells. In addition, neutralizing antibodies to IGF-II also inhibited the growth of these cells suggesting a potential autocrine function for this cytokine. We also compared cultured AT/RT cells to established cell lines to identify consistent drug sensitivity patterns among these cells. In addition to previously described cell lines and xenograft models, continuous culture of CSF derived cells may also provide an effective way to study the biology of AT/RT and to identify potential targets for future therapeutics for this tumor.

The activation of ErbB tyrosine kinase receptors (ErbB1, -2, -3, and -4) by ligand-induced homo- or heterodimerization regulates cell growth, death, and differentiation. AG1478 and PD153035 (also know as AG1517) have been adopted as specific ErbB1 inhibitors based on their high specificity for ErbB1 as compared to ErbB2 in in vitro kinase assays. We compared their ability to inhibit ErbB receptor signaling in intact cells to that of a novel ErbB receptor kinase inhibitor, BIBX1382BS. Neither AG1478 nor PD153035 displayed any specificity for ErbB1-mediated signaling induced by transforming growth factor alpha (TGF-alpha) as compared to signaling initiated through the other ErbB kinases. In contrast, BIBX1382BS was more potent at inhibiting signaling induced by TGF-alpha than that induced by neuregulin1-beta1 or anti-ErbB2 agonist antibodies. Interestingly, this compound blocked antibody-induced ErbB4 homodimer activation at even lower concentrations than ErbB1-triggered signaling. Thus, BIBX1382BS, but not AG1478 and PD153035, can be employed to differentiate between the ErbB kinases in intact cells when used at appropriate concentrations.

ErbB receptors are important regulators of fetal organ development, including the fetal lung. They exhibit diversity in signaling potential, acting through homo- and heterodimers to cause different biological responses. We hypothesized that ErbB receptors show cell-specific and stimuli-specific activation, heterodimerization, and cellular localization patterns in fetal lung. We investigated this using immunoblotting, co-immunoprecipitation, and confocal microscopy in primary isolated E19 fetal rat lung fibroblasts and epithelial type II cells, stimulated with epidermal growth factor, transforming growth factor alpha, neuregulin 1beta, or treated with conditioned medium (CM) from the respective other cell type. Fetal type II cells expressed significantly more ErbB1, ErbB2, and ErbB3 protein than fibroblasts. ErbB4 was consistently identified by co-immunoprecipitation of all other ErbB receptors in both cell types independent of the treatments. Downregulation of ErbB4 in fibroblasts initiated cell-cell communication that stimulated surfactant phospholipid synthesis in type II cells. Confocal microscopy in type II cells revealed nuclear localization of all receptors, most prominently for ErbB4. Neuregulin treatment resulted in relocation to the extra-nuclear cytoplasmic region, which was distinct from fibroblast CM treatment which led to nuclear localization of ErbB4 and ErbB2, inducing co-localization of both receptors. We speculate that ErbB4 plays a prominent role in fetal lung mesenchyme-epithelial communication.

The Consortium on the Genetics of Schizophrenia Family Study (COGS-1) has previously reported our efforts to characterize the genetic architecture of 12 primary endophenotypes for schizophrenia. We now report the characterization of 13 additional measures derived from the same endophenotype test paradigms in the COGS-1 families. Nine of the measures were found to discriminate between schizophrenia patients and controls, were significantly heritable (31 to 62%), and were sufficiently independent of previously assessed endophenotypes, demonstrating utility as additional endophenotypes. Genotyping via a custom array of 1536 SNPs from 94 candidate genes identified associations for CTNNA2, ERBB4, GRID1, GRID2, GRIK3, GRIK4, GRIN2B, NOS1AP, NRG1, and RELN across multiple endophenotypes. An experiment-wide p value of 0.003 suggested that the associations across all SNPs and endophenotypes collectively exceeded chance. Linkage analyses performed using a genome-wide SNP array further identified significant or suggestive linkage for six of the candidate endophenotypes, with several genes of interest located beneath the linkage peaks (e.g., CSMD1, DISC1, DLGAP2, GRIK2, GRIN3A, and SLC6A3). While the partial convergence of the association and linkage likely reflects differences in density of gene coverage provided by the distinct genotyping platforms, it is also likely an indication of the differential contribution of rare and common variants for some genes and methodological differences in detection ability. Still, many of the genes implicated by COGS through endophenotypes have been identified by independent studies of common, rare, and de novo variation in schizophrenia, all converging on a functional genetic network related to glutamatergic neurotransmission that warrants further investigation.

Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs). We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo), to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

Maternal immune activation (MIA) is an environmental risk factor for schizophrenia, and may contribute to other developmental disorders including autism and epilepsy. Activation of pro-inflammatory cytokine systems by injection of the synthetic double-stranded RNA polyriboinosinic-polyribocytidilic acid (Poly I:C) mediates important neurochemical and behavioral corollaries of MIA, which have relevance to deficits observed in schizophrenia. We examined the consequences of MIA on forebrain expression of neuregulin-1 (NRG-1), brain-derived neurotrophic factor (BDNF) and their receptors, ErbB4 and trkB, respectively, genes associated with schizophrenia. On gestational day 14, pregnant rats were injected with Poly I:C or vehicle. Utilizing in situ hybridization, expression of NRG-1, ErbB4, BDNF, and trkB was examined in male rat offspring at postnatal day (P) 14, P30 and P60. ErbB4 mRNA expression was significantly increased at P30 in the anterior cingulate (AC Ctx), frontal, and parietal cortices, with increases in AC Ctx expression continuing through P60. ErbB4 expression was also elevated in the prefrontal cortex (PFC) at P14. In contrast, NRG-1 mRNA was decreased in the PFC at P60. Expression of BDNF mRNA was significantly upregulated in the PFC at P60 and decreased in the AC Ctx at P14. Expression of trkB was increased in two regions, the piriform cortex at P14 and the striatum at P60. These findings demonstrate developmentally and regionally selective alterations in the expression of schizophrenia-related genes as a consequence of MIA. Further study is needed to determine contributions of these effects to the development of alterations of relevance to neuropsychiatric diseases.

Clear cell sarcoma of soft tissue (CCSST) represents a highly malignant tumor of the musculoskeletal system that is characterized by the chromosomal translocation t(12;22)(q13;q12) of the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1). In a former microarray expression study, we identified ERBB3, a member of the epidermal growth factor receptor (EGFR) family, as a promising new diagnostic marker in the differential diagnosis of CCSST. Here we show that, besides ErbB3, all CCSST cell lines (n = 8) also express the ErbB2 receptor or the ErbB4 receptor, representing an adequate coreceptor of ErbB3. The phosphorylation status of ErbB3 revealed these receptor pairs to be either constitutively activated in CCSST cells with high neuregulin-1 (NRG1) expression (n = 4) or activatable by exogenic NRG1 in cells showing low amounts of NRG1 mRNA (n = 4). Exogenous NRG1 stimulated the growth of a subset of CCSST cells but did not affect the kinetics of another subset. This difference was not strictly dependent on endogenous NRG1 expression; however, the growth-inhibiting effect of the pan-ErbB tyrosine kinase inhibitor CI-1033 or PD158780 clearly correlated with NRG1 expression indicating an autocrine growth stimulation loop which may constitute an interesting target of new therapeutic strategies in this tumor entity.

We have previously demonstrated that the constitutively active Q646C mutant of the ErbB4 receptor tyrosine kinase inhibits colony formation by human prostate tumor cell lines. Here we use ErbB4 mutants to identify ErbB4 functions critical for inhibiting colony formation. A derivative of ErbB4 Q646 that lacks kinase activity fails to inhibit colony formation by prostate tumor cells. Likewise, an ErbB4 Q646C mutant in the context of the CT-b splicing isoform fails to inhibit colony formation. Mutation of tyrosine 1056 to phenylalanine abrogates inhibition of colony formation whereas an ErbB4 mutant that lacks all of the putative sites of tyrosine phosphorylation except for tyrosine 1056 still inhibits colony formation. Given that tyrosine 1056 is missing in the CT-b isoform, these results suggest that phosphorylation of tyrosine 1056 is critical for function. Indeed, an ErbB4 mutant that lacks kinase activity but has a glutamate phosphomimic residue substituted for tyrosine 1056 inhibits colony formation. Finally, 1-dimensional phosphopeptide mapping indicates that ErbB4 Q646C is phosphorylated on tyrosine 1056. These data suggest that phosphorylation of ErbB4 tyrosine 1056 is critical for coupling ErbB4 to prostate tumor suppression.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as an in vitro model system in cardiac biology and in drug discovery (e.g., cardiotoxicity testing). Qualification of these cells for use in mechanistic investigations will require detailed evaluations of cardiomyocyte signaling pathways and cellular responses. ErbB signaling and the ligand neuregulin play critical roles in survival and functional integrity of cardiac myocytes. As such, we sought to characterize the expression and activity of the ErbB family of receptors. Antibody microarray analysis performed on cell lysates derived from maturing hiPSC-CMs detected expression of ∼570 signaling proteins. EGFR/ErbB1, HER2/ErbB2, and ErbB4, but not ErbB3 receptors, of the epidermal growth factor receptor family were confirmed by Western blot. Activation of ErbB signaling by neuregulin-1β (NRG, a natural ligand for ErbB4) and its modulation by trastuzumab (a monoclonal anti-ErbB2 antibody) and lapatinib (a small molecule ErbB2 tyrosine kinase inhibitor) were evaluated through assessing phosphorylation of AKT and Erk1/2, two major downstream kinases of ErbB signaling, using nanofluidic proteomic immunoassay. Downregulation of ErbB2 expression by siRNA silencing attenuated NRG-induced AKT and Erk1/2 phosphorylation. Activation of ErbB signaling with NRG, or inhibition with trastuzumab, alleviated or aggravated doxorubicin-induced cardiomyocyte damage, respectively, as assessed by a real-time cellular impedance analysis and ATP measurement. Collectively, these results support the expanded use of hiPSC-CMs to examine mechanisms of cardiotoxicity and support the value of using these cells in early assessments of cardiotoxicity or efficacy.

The current consensus recognizes four main medulloblastoma subgroups (wingless, Sonic hedgehog, group 3 and group 4). While medulloblastoma subgroups have been characterized extensively at the (epi-)genomic and transcriptomic levels, the proteome and phosphoproteome landscape remain to be comprehensively elucidated. Using quantitative (phospho)-proteomics in primary human medulloblastomas, we unravel distinct posttranscriptional regulation leading to highly divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas. Specifically, proteomic and phosphoproteomic analyses identify aberrant ERBB4-SRC signaling in group 4. Hence, enforced expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 medulloblastoma. Therefore, our integrative proteogenomics approach unveils an oncogenic pathway and potential therapeutic vulnerability in the most common medulloblastoma subgroup.

ErbB4 is a receptor tyrosine kinase implicated in the development and homeostasis of the heart, central nervous system, and mammary gland. Cleavable isoforms of ErbB4 release a soluble intracellular domain (ICD) that can translocate to the nucleus and function as a transcriptional coregulator. In search of regulatory mechanisms of ErbB4 ICD function, we identified PIAS3 as a novel interaction partner of ErbB4 ICD. In keeping with the small ubiquitin-like modifier (SUMO) E3 ligase function of protein inhibitor of activated STAT (PIAS) proteins, we showed that the ErbB4 ICD is modified by SUMO, and that PIAS3 stimulates the SUMOylation. Upon overexpression of PIAS3, the ErbB4 ICD generated from the full-length receptor accumulated into the nucleus in a manner that was dependent on the functional nuclear localization signal of ErbB4. In the nucleus, ErbB4 colocalized with PIAS3 and SUMO-1 in promyelocytic leukemia nuclear bodies, nuclear domains involved in regulation of transcription. Accordingly, PIAS3 overexpression had an effect on the transcriptional coregulatory activity of ErbB4, repressing its ability to coactivate transcription with Yes-associated protein. Finally, knockdown of PIAS3 with siRNA partially rescued the inhibitory effect of the ErbB4 ICD on differentiation of MDA-MB-468 breast cancer and HC11 mammary epithelial cells. Our findings illustrate that PIAS3 is a novel regulator of ErbB4 receptor tyrosine kinase, controlling its nuclear sequestration and function.

The erbB2 oncogene belongs to the type I trans-membrane tyrosine kinase family of receptors. Its medical importance stems from its widespread over-expression in breast cancer. This review will focus on the signal transduction through this protein, and explains how the overexpression of erbB2 may result in poor prognosis of breast cancer, and finally it will summerize our current understanding about the therapeutic potential of receptor-targeted therapy in breast cancer. ErbB2 does not have any known ligand which is able to bind to it with high affinity. However the kinase activity of erbB2 can be activated without any ligand, if it is overexpressed, and by heteroassociation with other members of the erbB family (erbB1 or epidermal growth factor receptor, erbB3 and erbB4). This interaction substantially increases the efficiency and diversity of signal transduction through these receptor complexes. In addition, erbB2 forms large scale receptor clusters containing hundreds of proteins. These receptor islands may take part in recruiting cytosolic factors which relay the signal towards the nucleus or the cytoplasm. Overexpression of erbB2 was linked to higher transforming activity, increased metastatic potential, angiogenesis and drug resistence of breast tumor in laboratory experiments. As a corollary of these properties, erbB2 amplification is generally thought to be associated with a poor prognosis in breast cancer patients. These early findings lead to the development of antibodies that down-regulate erbB2. Such a therapeutic approach has already been found effective in experimental tumor models and in clinical trials as well. Further understanding of the importance of erbB2 and growth factor receptors in the transformation of normal cells to malignant ones may once give us a chance to cure erbB2 over-expressing breast cancer.

Many studies correlating gene expression data to clinical parameters assume a linear increase or decrease of the clinical parameter under investigation with the expression of a gene. We have studied genes encoding important breast cancer-related proteins using a model for survival-type data that is based on natural splines and the Cox proportional hazard model, thereby removing the linearity assumption. Expression data of 16 genes were studied in relation to metastasis-free probability in a cohort of 295 consecutive breast cancer patients treated at The Netherlands Cancer Institute. The independent predictive power for disease outcome of the 16 individual genes was tested in a multivariable model with known clinical and pathological risk factors. There is a linear relationship between increasing expression and a higher or lower hazard for distant metastasis for ESR1, ERBB4, VEGF, CCNE2, EZH2, and UPA; for ERBB2, ERBB3, CCND1, CCNE1, EED, CXCR4, CCR7, SDF1, and PAI1 there is no clear increase or decrease; and for EGFR there seems to be a non-linear relation. Multivariable analysis showed that the 70-gene prognosis profile outperforms all the other variables in the model (hazard-rate 5.4, 95% CI 2.5-11.7; P = 0.000018). EGFR-expression seems to have a non-linear relation with disease outcome, indicating that lower but also higher expression of EGFR are associated with worse outcome compared to intermediate expression levels; the other genes show no or a linear relation.