OBJECTIVE

Assessment of expression levels of Wwox, Wwox-interacting proteins Ap2alpha, Ap2gamma, and ErbB4, the Ap2gamma transcriptional target protein Her2, and the possible Ap2alpha transcriptional target PrkaRIalpha, in breast cancers, to determine their roles in tamoxifen resistance. The hypothesis was that sequestration of Wwox interactors in the cytoplasm might control tamoxifen response.

METHODS

Tissue sections from 51 tamoxifen-sensitive and 38 tamoxifen-resistant, estrogen receptor alpha-positive breast cancers were stained for the above proteins, as well as progesterone receptor (PR). The relation of tamoxifen resistance and other clinical features, with level of expression of these proteins, and pairwise correlations among various immunohistochemical markers were determined.

RESULTS

Menopausal status, tumor, node, and stage, loss of PR, lost or reduced expression of Wwox, and high level of expression of PrkaRIalpha, Ap2gamma, and Her2 were significantly correlated with tamoxifen resistance. In multivariate analysis, Wwox, PrkaRIalpha, Ap2gamma, and ErbB4 were found to be independent markers of tamoxifen resistance. Reduced Wwox expression was better than PR in prediction of resistance, especially in high-risk patients, and nuclear Ap2gamma expression was better than Her2, especially in low-risk patients.

CONCLUSIONS

The results illustrate the complex relationships among the marker proteins assessed in this in vivo study and suggest new markers for prediction of response to tamoxifen treatment as well as possible new targets for treatment of breast cancer. Wwox and Ap2gamma emerge as new biomarkers that may be superior to PR and Her2 in predicting tamoxifen response.

Neuregulin-1 (NRG1) signaling is thought to contribute to both neuronal development and schizophrenia neuropathology. Here, we describe the developmental effects of excessive peripheral NRG1 signals on synaptic activity and AMPA receptor expression of GABAergic interneurons in postnatal rodent neocortex. A core peptide common to all NRG1 variants (eNRG1) was subcutaneously administered to mouse pups. Injected eNRG1 penetrated the blood-brain barrier and activated ErbB4 NRG1 receptors in the neocortex, in which ErbB4 mRNA is predominantly expressed by parvalbumin-positive GABAergic interneurons. We prepared neocortical slices from juvenile mice that were receiving eNRG1 subchronically and recorded inhibitory synaptic activity from layer V pyramidal neurons. Postnatal eNRG1 treatment significantly enhanced polysynaptic IPSCs, although monosynaptic IPSCs were not affected. Examination of excitatory inputs to parvalbumin-containing GABAergic interneurons revealed that eNRG1 treatment significantly increased AMPA-triggered inward currents and the amplitudes and frequencies of miniature EPSCs (mEPSCs). Similar effects on mEPSCs were observed in mice treated with a soluble, full-length form of NRG1 type I. Consistent with the electrophysiologic data, expression of the AMPA receptor GluA1 (i.e., GluR1, GluRA) was upregulated in the postsynaptic density/cytoskeletal fraction prepared from eNRG1-treated mouse neocortices. Cortical GABAergic neurons cultured with eNRG1 exhibited a significant increase in surface GluA1 immunoreactivity at putative synaptic sites on their dendrites. These results indicate that NRG1 circulating in the periphery influences postnatal development of synaptic AMPA receptor expression in cortical GABAergic interneurons and may play a role in conditions characterized by GABA-associated neuropathologic processes.

Cleavable isoforms of the ErbB4 receptor tyrosine kinase release a soluble intracellular domain (ICD) that may translocate to the nucleus and regulate signaling. However, ErbB4 gene is alternatively spliced generating CYT-1 and CYT-2 isoforms with different cytoplasmic tails. Here, we addressed whether the two alternative ErbB4 ICDs of either CYT-1 (ICD1) or CYT-2 (ICD2) type differ in signaling to the nucleus. Confocal microscopy and extraction of nuclear cell fractions indicated that significantly more ICD2 translocated to the nuclei when compared to ICD1. Unlike the membrane-anchored 80 kDa fragments derived from full-length ErbB4 isoforms, the two ICDs did not differ from each other in metabolic stability or ubiquitylation. However, ICD2 was phosphorylated at tyrosine residues to a higher extent and demonstrated greater in vitro kinase activity than ICD1. Mutating the ATP-binding site within ICD2 kinase domain (ICD2 K751R) blocked its tyrosine phosphorylation and significantly reduced its nuclear translocation. When expressed in the context of full-length ErbB4, ICD2 was also more efficient than ICD1 in promoting transcriptional activation of the STAT5 target gene beta-casein. These findings indicate that the two alternative ICDs of ErbB4 differ in their nuclear accumulation, and that the mechanism involves differential kinase activity but not ubiquitin-regulated ICD stability.

Heregulin-mediated activation of particular erbB receptor combinations was used as a model system to investigate the interaction of erbB3 and erbB4 with the adaptor protein growth factor receptor-bound (Grb)7. In human breast cancer cell lines, co-immunoprecipitation of Grb7 with both receptors was detected upon heregulin stimulation. This association was direct and mediated by the Grb7 Src homology (SH)2 domain. Co-expression of erbB2 with erbB3 point mutants was used to map Grb7 binding sites. This demonstrated that tyrosine 1180 and 1243 represent the major and minor sites of Grb7 interaction, respectively. Although these recognition sequences possess an Asn residue at +2 relative to the phosphotyrosine and therefore represent potential Grb2 binding sites, phosphopeptide competition and "pull-down" experiments demonstrated that they interact preferentially with the Grb7 versus the Grb2 SH2 domain. Substitution analysis indicated that an Arg residue at +3 could act as a selectivity determinant, but the effect was context-dependent. Consequently, the Grb2 and Grb7 SH2 domains possess overlapping, but distinct, specificities. These studies therefore identify Grb7 as an in vivo target of erbB3 and erbB4 and provide an underlying mechanism for the ability of erbB3 to recruit Grb7 and not Grb2, a property unique among erbB receptors.

The development of pancreatic fibrosis has been shown to be a major component in several diseases of the pancreas including pancreatic cancer, chronic pancreatitis, and type 2 diabetes mellitus, but its actual role in the progression of these disorders is still unknown. This fibrosis is characterized by stromal expansion and the excessive deposition of extracellular matrix (ECM) that replaces pancreatic tissue. This eventually leads to dysregulation of ECM turnover, production of cytokines, restriction of blood flow, and often exocrine and endocrine insufficiencies. Activated pancreatic stellate cells (PSCs) have been identified as key mediators in the progression of pancreatic fibrosis, serving as the predominant source of excess ECM proteins. Previously, we found that overexpression of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pancreatic islets led to intraislet fibrosis. HB-EGF binds to and activates two receptors, epidermal growth factor receptor (EGFR) and ErbB4, as well as heparin moieties and CD9/DRAP27. To understand the mechanism underlying the induction of fibrogenesis by HB-EGF, we utilized a hypomorphic allele of Egfr, the Waved-2 allele, to demonstrate that EGFR signaling regulates fibrogenesis in vivo. Using an in vitro cell migration assay, we show that HB-EGF regulates both chemoattraction and stimulation of proliferation of PSCs via EGFR activation.

Tumor progression is intrinsically tied to the clonal selection of tumor cells with acquired phenotypes allowing to cope with a hostile microenvironment. Hypoxia-inducible factors (HIFs) master the transcriptional response to local tissue hypoxia, a hallmark of solid tumors. Here, we report significantly longer patient survival in breast cancer with high levels of HIF-2α. Amphiregulin (AREG) and WNT1-inducible signaling pathway protein-2 (WISP2) expression was strongly HIF-2α-dependent and their promoters were particularly responsive to HIF-2α. The endogenous AREG promoter recruited HIF-2α in the absence of a classical HIF-DNA interaction motif, revealing a novel mechanism of gene regulation. Loss of AREG expression in HIF-2α-depleted cells was accompanied by reduced activation of epidermal growth factor (EGF) receptor family members. Apparently opposing results from patient and in vitro data point to an HIF-2α-dependent auto-stimulatory tumor phenotype that, while promoting EGF signaling in cellular models, increased the survival of diagnosed and treated human patients. Our findings suggest a model where HIF-2α-mediated autocrine growth signaling in breast cancer sustains a state of cellular self-sufficiency, thereby masking unfavorable microenvironmental growth conditions, limiting adverse selection and improving therapy efficacy. Importantly, HIF-2α/AREG/WISP2-expressing tumors were associated with luminal tumor differentiation, indicative of a better response to classical treatments. Shifting the HIF-1/2α balance toward an HIF-2-dominated phenotype could thus offer a novel approach in breast cancer therapy.

The first three members of the ErbB family of receptor tyrosine kinases activate a wide variety of signaling pathways and are frequently misregulated in cancer. Much less is known about ErbB4. Here we use tandem mass spectrometry to identify 19 sites of tyrosine phosphorylation on ErbB4, and protein microarrays to quantify biophysical interactions between these sites and virtually every SH2 and PTB domain encoded in the human genome. Our unbiased approach highlighted several previously unrecognized interactions and led to the finding that ErbB4 can recruit and activate STAT1. At a systems level, we found that ErbB4 is much more selective than the other ErbB receptors. This suggests that ErbB4 may enable ErbB2 and ErbB3 to signal independently of EGFR under normal conditions, and provides a possible explanation for the protective properties of ErbB4 in cancer.

The neuregulin (NRG)/epidermal growth factor (EGF) family of growth factors consists of several ligands that specifically activate four erbB receptor-tyrosine kinases, namely erbB-1 (EGF-R), erbB-2 (neu), erbB-3, and erbB-4. We have previously shown that islet morphogenesis is impaired and beta-cell differentiation delayed in mice lacking functional EGF-R [EGF-R (-/-)]. The present study aims to clarify which erbB ligands are important for islet development. Pancreatic expression of EGF, TGF-alpha, heparin-binding EGF, betacellulin (BTC), and NRG-4 was detected as early as embryonic d 13 (E13). Effects of these ligands were studied in E12.5 pancreatic explant cultures grown for 5 d ex vivo. None of the growth factors affected the ratio of endocrine to exocrine cells. However, significant effects within the endocrine cell populations were induced by EGF, BTC, and NRG-4. beta-Cell development was augmented by BTC, whereas the development of somatostatin-expressing delta-cells was stimulated by NRG-4. Both ligands decreased the numbers of glucagon-containing alpha-cells. The effect of BTC was abolished in the EGF-R (-/-) mice. A soluble erbB-4 binding fusion protein totally inhibited the effects of NRG-4 but not of BTC. Neutralization of endogenous NRG-4 activity in the model system effectively inhibited delta-cell development, indicating that this erbB4-ligand is an essential factor for delineation of the somatostatin-producing delta-cells. Our results suggest that ligands of the EGF-R/erbB-1 and erbB-4 receptors regulate the lineage determination of islet cells during pancreatic development. BTC, acting through EGF-R/erbB-1, is important for the differentiation of beta-cells. This could be applied in the targeted differentiation of stem cells into insulin-producing cells.

Although epidermal growth factor (EGF) and neuregulin-1 are neurotrophic factors for mesencephalic dopaminergic neurons and implicated in schizophrenia, the cellular localization and developmental regulation of their receptors (ErbB1-4) remain to be characterized. Here we investigated the distributions of mRNA for ErbB1-4 in the midbrain of the developing mouse with in situ hybridization and immunohistochemistry. The expression of ErbB1 and ErbB2 mRNAs was relatively high at the perinatal stage and frequently colocalized with mRNA for S100beta and Olig2, markers for immature astrocytes or oligodendrocyte precursors. Modest signal for ErbB1 mRNA was also detected in a subset of dopaminergic neurons. ErbB3 mRNA was detectable at postnatal day 10, peaked at postnatal day 18, and colocalized with 2',3'-cyclic nucleotide 3'-phosphodiesterase, a marker for oligodendrocytes. In contrast, ErbB4 mRNA was exclusively localized in neurons throughout development. Almost all of ErbB4 mRNA-expressing cells (94%-96%) were positive for tyrosine hydroxylase in the substantia nigra pars compacta but 66%-78% in the ventral tegmental area and substantia nigra pars lateralis. Conversely, 92%-99% of tyrosine hydroxylase-positive cells expressed ErbB4 mRNA. The robust and restricted expression of ErbB4 mRNA in the midbrain dopaminergic neurons suggests that ErbB4 ligands, neuregulin-1 and other EGF-related molecules, contribute to development or maintenance of this neuronal population.

OBJECTIVE

The protein expression and interactions of the ErbB receptors were examined in different liver proliferation models in vivo and in vitro, including ontogeny and regeneration following partial hepatectomy.

METHODS

Expression and tyrosine phosphorylation status of specific ErbB proteins were studied by immunologic methods.

RESULTS

The epidermal growth factor receptor, ErbB2, and ErbB3 were the only ErbB proteins detected in the liver parenchyma on embryonic day 19. ErbB2 disappeared by the third week after birth and could not be appreciably induced in the adult animal by partial hepatectomy. ErbB2 was also detected in multipotent stem (RLE) and hepatoma (H4IIe) cell lines as well as in fetal, but not adult, hepatocyte cultures. Only epidermal growth factor receptor and ErbB3 were detected in adult liver, and both showed circadian variation in protein expression. ErbB4 was not detected in any model. Patterns of ligand-induced ErbB phosphorylation differed between fetal and adult hepatocytes.

CONCLUSIONS

Complex and independent programs regulate the ErbB receptors, with implications for differential cell signaling in hepatic development and regeneration.

Prenatal stress has been widely demonstrated to have links with behavioral problems in clinical populations and animal models, however, few investigations have examined the immediate developmental events that are affected by prenatal stress. Here, we utilize GAD67GFP transgenic mice in which GABAergic progenitors express green fluorescent protein (GFP) to examine the impact of prenatal stress on the development of these precursors to inhibitory neurons. Pregnant female mice were exposed to restraint stress three times daily from embryonic day 12 (E12) onwards. Their offspring demonstrated changes in the distribution of GFP-positive (GFP+) GABAergic progenitors in the telencephalon as early as E13 and persisting until postnatal day 0. Changes in distribution reflected alterations in tangential migration and radial integration of GFP+ cells into the developing cortical plate. Fate mapping of GAD67GFP+ progenitors with bromodeoxyuridine injected at E13 demonstrated a significant increase of these cells at P0 in anterior white matter. An overall decrease in GAD67GFP+ progenitors at P0 in medial frontal cortex could not be attributed to a reduction in cell proliferation. Significant changes in dlx2, nkx2.1 and their downstream target erbb4, transcription factors which regulate interneuron migration, were found within the prenatally stressed developing forebrain, while no differences were seen in mash1, a determinant of interneuron fate, bdnf, a maturation factor for GABAergic cells or fgf2, an early growth/differentiation factor. These results demonstrate that early disruption in GABAergic progenitor migration caused by prenatal stress may be responsible for neuronal defects in disorders with GABAergic abnormalities like schizophrenia.

Afatinib (also known as BIBW 2992) has recently been approved in several countries for the treatment of a distinct type of epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer. This manuscript comprehensively reviews the preclinical data on afatinib, an irreversible inhibitor of the tyrosine kinase activity of members of the epidermal growth factor receptor family (ErbB) including EGFR, HER2 and ErbB4. Afatinib covalently binds to cysteine 797 of the EGFR and the corresponding cysteines 805 and 803 in HER2 and ErbB4, respectively. Such covalent binding irreversibly inhibits the tyrosine kinase activity of these receptors, resulting in reduced auto- and transphosphorylation within the ErbB dimers and inhibition of important steps in the signal transduction of all ErbB receptor family members. Afatinib inhibits cellular growth and induces apoptosis in a wide range of cells representative for non-small cell lung cancer, breast cancer, pancreatic cancer, colorectal cancer, head and neck squamous cell cancer and several other cancer types exhibiting abnormalities of the ErbB network. This translates into tumour shrinkage in a variety of in vivo rodent models of such cancers. Afatinib retains inhibitory effects on signal transduction and in vitro and in vivo cancer cell growth in tumours resistant to reversible EGFR inhibitors, such as those exhibiting the T790M mutations. Several combination treatments have been explored to prevent and/or overcome development of resistance to afatinib, the most promising being those with EGFR- or HER2-targeted antibodies, other tyrosine kinase inhibitors or inhibitors of downstream signalling molecules.

Recently identified candidate susceptibility genes for schizophrenia are likely to play, important roles in the pathophysiology of the illness. It is also clear, however, that the etiologic, contribution of these genes is not only via their own functions but also through interactions with other, genes and environmental factors. Genetic, transgenic and postmortem brain studies support a, potential role for NRG1-erbB4 signaling in schizophrenia. Embedded in the results of these studies, however, are clues to the notion that NRG1-erbB4 signaling does not act alone but in conjunction with, other pathways. This article aims to re-evaluate the evidence for the role of neuregulin 1 (NRG1)-erbB4 signaling in schizophrenia by focusing on its interactions with other candidate susceptibility, pathways. In addition, we consider molecular substrates upon which the NRG1-erbB4 and other, candidate pathways converge contributing to susceptibility for the illness (schizophrenia interactome). Glutamatergic signaling can be an interesting candidate for schizophrenia interactome. Schizophrenia is associated with NMDA receptor hypofunction and moreover, several susceptibility genes for, schizophrenia converge on NMDA receptor signaling. These candidate genes influence NMDA receptor, signaling via diverse mechanisms, yet all eventually impact on protein composition of NMDA receptor, complexes. Likewise, the protein associations in the receptor complexes can themselves modulate, signaling molecules of candidate genes and their pathways. Therefore, protein-protein interactions in the NMDA receptor complexes can mediate reciprocal interactions between NMDA receptor function, and susceptibility candidate pathways including NRG1-erbB4 signaling and thus can be a, schizophrenia interactome.

To investigate the role of neuron-glial cell interactions in the auditory nerve, we asked whether spiral ganglion neurons (SGNs) express neuregulin and whether neuregulin regulates proliferation and/or neurotrophin expression in spiral ganglion Schwann cells (SGSCs). Using immunocytochemistry, we found that type I and type II SGNs express neuregulin in vivo and in vitro. Cultured SGSCs express the neuregulin receptors ErbB2 and ErbB3, but not ErbB4. Neuregulin activates ErbB2 and ErbB3 in cultured SGSCs, evidenced by increased tyrosine phosphorylation of the receptors following neuregulin treatment. Neuregulin treatment increased the proliferation rate of cultured SGSCs by 2.5-fold. Fibroblast growth factor-2 (FGF-2) and transforming growth factor beta (TGF-beta) also increased SGSC proliferation. The mitogenic effect of neuregulin and FGF-2 was blocked by inhibition of mitogen-activated protein kinase signaling but not by inhibition of phosphatidylinositol-3'-OH kinase. Using RT-PCR, we found that cultured SGSCs express neurotrophins, including brain-derived neurotrophic factor and neurotrophin-3 (NT-3), raising the possibility that SGSCs contribute to the trophic support of SGNs. Treatment with neither neuregulin nor TGF-beta increased neurotrophin expression in cultured SGSCs, as had been observed in developing sympathetic ganglia, but appeared to negatively regulate NT-3 expression. Thus, neuregulin and neurotrophins may mediate reciprocal neuron-glial interactions in the auditory nerve.

OBJECTIVE

To compare the immunohistochemically assessed expression of the epidermal growth factor receptor (EGFR) family in normal and malignant bladder urothelium, and suggest new hypotheses about their function in the development and progression of transitional cell carcinoma (TCC).

METHODS

EGFR, ERBB2, ERBB3 and ERBB4 were evaluated immunohistochemically in normal urothelium (NU, 15), primary non-metastasized invasive TCC (NMC, 19) and in primary invasive TCCs with corresponding metastases (MC, 51, both specimens).

RESULTS

All NU samples expressed ERBB4, none expressed ERBB2 and two expressed EGFR; all staining was uniform throughout all cell layers. ERBB2 expression increased and ERBB4 decreased from normal samples to carcinomas. There was no difference between NMCs and MCs in ERBB2, ERBB3 and ERBB4, but the NMCs expressed more EGFR than both NU and MC samples. There were no associations with T category, grade or survival. All combinations of expression levels for the four receptors were detected, with no dominant profile.

CONCLUSIONS

We hypothesise that: (i) ERBB4 is important for differentiation in NU; (ii) ERBB2 is up-regulated with carcinogenesis in the urinary bladder but does not discriminate between bladder cancer with or without metastases; (iii) EGFR may be a marker of indolent disease. A current hypothesis, that superficial layers of NU do not express EGFR and thus protect the basal cells from the mitogenic effect of urinary EGF, is challenged.

The receptor tyrosine kinase ERBB4, a member of the epidermal growth factor receptor (EGFR) family, is unusual in that ERBB4 can undergo intramembrane proteolysis, releasing a soluble intracellular domain (ICD) that modulates transcription in the nucleus. We found that ERBB4 activated the transcriptional coactivator YAP, which promotes organ and tissue growth and is inhibited by the Hippo tumor-suppressor pathway. Overexpressing ERBB4 in cultured mammary epithelial cells or adding the ERBB4 ligand neuregulin 1 (NRG1) to breast cancer cell cultures promoted the expression of genes regulated by YAP, such as CTGF. Knocking down YAP or ERBB4 prevented the induction of CTGF expression by NRG1, as did treating cells with the ERBB inhibitors lapatinib or erlotinib, which reduced ERBB4 cleavage. NRG1 stimulated YAP activity to an extent comparable to that of EGF (epidermal growth factor) or LPA (lysophosphatidic acid), known activators of YAP. NRG1 stimulated YAP-dependent cell migration in breast cancer cell lines. These observations connect the unusual nuclear function of a growth factor receptor with a mechanosensory pathway and suggest that NRG1-ERBB4-YAP signaling contributes to the aggressive behavior of tumor cells.

The paralogous multifunctional adaptor proteins YAP and TAZ are the nuclear effectors of the Hippo pathway, a central mechanism of organ size control and stem cell self-renewal. WW domains, mediators of protein-protein interactions, are essential for YAP and TAZ function, enabling interactions with PPxY motifs of numerous partner proteins. YAP has single and double WW domain isoforms (YAP1 and YAP2) whereas only a single WW domain isoform of TAZ has been described to date. Here we identify the first example of a double WW domain isoform of TAZ. Using NMR, we have characterized conformational features and peptide binding of YAP and TAZ tandem WW domains (WW1-WW2). The solution structure of YAP WW2 confirms that it has a canonical three-stranded antiparallel β-sheet WW domain fold. While chemical shift-based analysis indicates that the WW domains in the tandem WW pairs retain the characteristic WW domain fold, 15N relaxation data show that, within the respective WW pairs, YAP WW1 and both WW1 and WW2 of TAZ undergo conformational exchange. 15N relaxation data also indicate that the linker between the WW domains is flexible in both YAP and TAZ. Within both YAP and TAZ tandem WW pairs, WW1 and WW2 bind single PPxY-containing peptide ligand concurrently and noncooperatively with sub-mM affinity. YAP and TAZ WW1-WW2 bind a dual PPxY-containing peptide with approximately 6-fold higher affinity. Our results indicate that both WW domains in YAP and TAZ are functional and capable of enhanced affinity binding to multi-PPxY partner proteins such as LATS1, ErbB4, and AMOT.

Next generation sequencing (NGS) has been used to characterize the overall genomic landscape of melanomas. Here, we systematically examined mutations from recently published melanoma NGS data involving 241 paired tumor-normal samples to identify potentially clinically relevant mutations. Melanomas were characterized according to an in-house clinical assay that identifies well-known specific recurrent mutations in five driver genes: BRAF (affecting V600), NRAS (G12, G13, and Q61), KIT (W557, V559, L576, K642, and D816), GNAQ (Q209), and GNA11 (Q209). Tumors with none of these mutations are termed "pan negative." We then mined the driver mutation-positive and pan-negative melanoma NGS data for mutations in 632 cancer genes that could influence existing or emerging targeted therapies. First, we uncovered several genes whose mutations were more likely associated with BRAF- or NRAS-driven melanomas, including TP53 and COL1A1 with BRAF, and PPP6C, KALRN, PIK3R4, TRPM6, GUCY2C, and PRKAA2 with NRAS. Second, we found that the 69 "pan-negative" melanoma genomes harbored alternate infrequent mutations in the five known driver genes along with many mutations in genes encoding guanine nucleotide binding protein α-subunits. Third, we identified 12 significantly mutated genes in "pan-negative" samples (ALK, STK31, DGKI, RAC1, EPHA4, ADAMTS18, EPHA7, ERBB4, TAF1L, NF1, SYK, and KDR), including five genes (RAC1, ADAMTS18, EPHA7, TAF1L, and NF1) with a recurrent mutation in at least two "pan-negative" tumor samples. This meta-analysis provides a road map for the study of additional potentially actionable genes in both driver mutation-positive and pan-negative melanomas.

Blastocyst implantation requires molecular and cellular interactions between the uterine luminal epithelium and blastocyst trophectoderm. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is induced in the mouse luminal epithelium solely at the site of blastocyst apposition at 16:00 hours on day 4 of pregnancy prior to the attachment reaction (22:00-23:00 hours), and that HB-EGF promotes blastocyst growth, zona-hatching and trophoblast outgrowth. To delineate which EGF receptors participate in blastocyst activation, the toxicity of chimeric toxins composed of HB-EGF or TGF-(&agr;) coupled to Pseudomonas exotoxin (PE) were used as measures of receptor expression. TGF-(&agr;) or HB-EGF binds to EGF-receptor (ErbB1), while HB-EGF, in addition, binds to ErbB4. The results indicate that ErbB1 is inefficient in mediating TGF-(&agr;)-PE or HB-EGF-PE toxicity as follows: (i) TGF-(&agr;)-PE was relatively inferior in killing blastocysts, 100-fold less than HB-EGF-PE, (ii) analysis of blastocysts isolated from cross-bred egfr+/- mice demonstrated that HB-EGF-PE, but not TGF-(&agr;)-PE, killed egfr-/- blastocysts, and (iii) blastocysts that survived TGF-(&agr;)-PE were nevertheless killed by HB-EGF-PE. HB-EGF-PE toxicity was partially mediated by cell surface heparan sulfate proteoglycans (HSPG), since a peptide corresponding to the heparin-binding domain of HB-EGF as well as heparitinase treatment protected the blastocysts from the toxic effects of HB-EGF-PE by about 40%. ErbB4 is a candidate for being an HB-EGF-responsive receptor since RT-PCR analysis demonstrated that day 4 mouse blastocysts express two different erbB4 isoforms and immunostaining with anti-ErbB4 antibodies confirmed that ErbB4 protein is expressed at the apical surface of the trophectoderm cells. It is concluded that (i) HB-EGF interacts with the blastocyst cell surface via high-affinity receptors other than ErbB1, (ii) the HB-EGF interaction with high-affinity blastocysts receptors is regulated by heparan sulfate, and (iii) ErbB4 is a candidate for being a high-affinity receptor for HB-EGF on the surface of implantation-competent blastocysts.

The ErbB family of receptors, which include the epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4 mediate the actions of a family of bioactive polypeptides. EGF signals through EGFR, whereas heregulin (HRG) signaling is initiated through binding to either ErbB3 or ErbB4. In this report we studied the role of protein-tyrosine phosphatase SHP-2 in ErbB-mediated activation of mitogen-activated protein kinase (MAPK) by overexpressing SHP-2 mutants in COS-7 cells. We demonstrate that enzymatic activity and both NH2- and COOH-terminal SH2 domains of SHP-2 are required for EGF-induced MAPK activation, but not for c-Jun amino-terminal kinase stimulation or MAPK activation which occurred in response to myristoylated son of sevenless, activated Ras, or phorbol ester. Dominant-negative forms of SHP-2 had no effect on EGF-stimulated interaction of GRB2 with EGFR or SHC, nor did they influence phosphorylation of SHC and SHC/EGFR association. The same mutant SHP-2 structures that inhibited EGF-mediated stimulation of MAPK also blocked HRG alpha/beta-induced MAPK activation. EGF or HRG beta caused SHP-2 SH2 domains to engage multiple phosphotyrosine proteins, and mutation of either domain disrupted these associations. These results demonstrate that SHP-2 performs a common and essential function(s) in ligand-stimulated MAPK activation by the ErbB family of receptors.

The role of receptor tyrosine kinases (RTKs) in transmembrane signaling is well established. Recently, ligand-dependent translocation of RTKs to the nucleus has been reported, but the functional importance of this process remains unclear. In this issue, Sardi et al. (2006) provide evidence for direct signaling in the nucleus by an intracellular ErbB4 receptor fragment that is released upon receptor activation by ligand. The fragment forms a complex with the adaptor TAB2 and the corepressor N-CoR and transits to the nucleus, where it represses transcription of genes that promote the formation of astrocytes.

There is a great unmet medical need in the area of cancer treatment. A potential therapeutic target for intervention in cancer is ADAM10. ADAM10 is a disintegrin-metalloproteinase that processes membrane bound proteins from the cell surface to yield soluble forms. Pharmaceutical companies are actively seeking out inhibitors of ADAM10 for treatments in cancer as the enzyme is known to release the ErbB receptor, HER2/ErbB2 from the cell membrane, an event that is necessary for HER2 positive tumor cells to proliferate. ADAM10 is also capable of processing betacellulin indicating that an inhibitor could be used against EGFR/ErbB1 and/or HER4/ErbB4 receptor positive tumor cells that are betacellulin-dependent. ADAM10 is the principle sheddase for several other molecules associated with cancer proliferation, differentiation, adhesion and migration such as Notch, E-cadherin, CD44 and L1 adhesion molecule indicating that targeting ADAM10 with specific inhibitors could be beneficial.

OBJECTIVE

Formalin-fixed, paraffin-embedded tumor samples from CALGB 80203 were analyzed for expression of EGFR axis-related genes to identify prognostic or predictive biomarkers for cetuximab treatment.

METHODS

Patients (238 total) with first-line metastatic colorectal cancer (mCRC) were randomized to FOLFOX or FOLFIRI chemotherapy ± cetuximab. qRT-PCR analyses were conducted on tissues from 103 patients at baseline to measure gene expression levels of HER-related genes, including amphiregulin (AREG), betacellulin (BTC), NT5E (CD73), DUSP4, EGF, EGFR, epigen (EPGN), epiregulin (EREG), HBEGF, ERBB2 (HER2), ERBB3 (HER3), ERBB4 (HER4), PHLDA1, and TGFA. The interactions between expression levels and treatment with respect to progression-free survival (PFS) and overall survival (OS) were modeled using multiplicative Cox proportional hazards models.

RESULTS

High tumor mRNA levels of HER2 [hazard ratio (HR), 0.64; P = 0.002] and EREG (HR, 0.89; P = 0.016) were prognostic markers associated with longer PFS across all patients. HER3 and CD73 expression levels were identified as potential predictive markers of benefit from cetuximab. In KRAS wild-type (WT) tumors, low HER3 expression was associated with longer OS from cetuximab treatment, whereas high HER3 expression was associated with shorter OS from cetuximab treatment (chemo + cetuximab: HR, 1.15; chemo-only: HR, 0.48; Pinteraction = 0.029). High CD73 expression was associated with longer PFS from cetuximab treatment in patients with KRAS-WT (chemo + cetuximab: HR, 0.91; chemo-only: HR, 1.57; Pinteraction = 0.026) and KRAS-mutant (Mut) tumors (chemo + cetuximab: HR, 0.80; chemo-only: HR, 1.29; P = 0.025).

CONCLUSIONS

Gene expression of HER3 and CD73 was identified as a potential predictive marker for cetuximab. These data implicate HER axis signaling and immune modulation as potential mechanisms of cetuximab action and sensitivity.

Ligands for ErbB1-4 receptor tyrosine kinases, such as epidermal growth factor (EGF) and neuregulins, regulate brain development and function. Thus, abnormalities in their signaling are implicated in the etiology or pathology of schizophrenia and Parkinson's disease. Among the ErbB receptors, ErbB1, and ErbB4 are expressed in dopamine and GABA neurons, while ErbB1, 2, and/or 3 are mainly present in oligodendrocytes, astrocytes, and their precursors. Thus, deficits in ErbB signaling might contribute to the neurological and psychiatric diseases stemming from these cell types. By incorporating the latest cancer molecular biology as well as our recent progress, we discuss signal cross talk between the ErbB1-4 subunits and their neurobiological functions in each cell type. The potential contribution of virus-derived cytokines (virokines) that mimic EGF and neuregulin-1 in brain diseases are also discussed.