We previously demonstrated that pancreatic adenocarcinoma BxPC-3 xenografts display resistance to treatment with Erbitux, gemcitabine, and radiation, whereas MIA PaCa-2 xenografts are highly sensitive to the same therapy. Here, we elucidate in vitro mechanisms that may explain the observed differential response of epidermal growth factor receptor (EGFR) expressing pancreatic adenocarcinoma xenografts to Erbitux-based combination therapy in vivo. MIA PaCa-2 and BxPC-3 protein lysates were probed with antibodies to EGFR, ErbB2, ErbB3, and ErbB4. Constitutive ErbB3 activity was visualized by immunoblot analysis using anti-phosphotyrosine antibodies and receptor-specific immunoprecipitates. erbB2 and erbB3 gene expression in both cell lines was quantified with real-time polymerase chain reaction. Erbitux-induced internalization of EGFR was determined by flow cytometry following Erbitux treatment for different incubation times at 0 degrees C and 37 degrees C. MIA PaCa-2 and BxPC-3 protein extracts were also probed with anti-phospho-mitogen-activated protein kinase antibody after stimulation with EGF and in the presence of Erbitux. Although both cell lines expressed EGFR and ErbB2 protein, ErbB3 protein was selectively expressed by BxPC-3 cells, where it also showed evidence of constitutive phosphorylation. There was a 10-fold increase of erbB3 transcript levels in BxPC-3 cells compared with MIA PaCa-2. ErbB4 protein was not detectable in either cell line. Erbitux mediated EGFR internalization in MIA PaCa-2 cells after 2 hours of incubation, whereas it did not promote EGFR internalization in BxPC-3 cells. Likewise, EGF-dependent phosphorylation of MAPK p44/42 was blocked by Erbitux treatment in MIA PaCa-2 but not BxPC-3 cells. Erbitux selectively interfered with EGF-induced MAPK activation in MIA PaCa-2 but not BxPC-3 cells. Persistent MAPK activation and impaired in vitro internalization of EGFR by BxPC-3 pancreatic cancer cells may be due to constitutive ErbB3 signaling, facilitated by heterodimerization with EGFR, which may explain resistance to Erbitux-based combination therapy in vivo.

We previously described spatiotemporal expression of various epidermal growth factor (EGF)-like ligands and receptor subtypes, ErbB1 and ErbB2, during the peri-implantation period. To better understand the roles of these ligands and their possible signaling schemes in implantation, it is important to define the status of all the ligands and receptor subtypes in the uterus/embryo. No information is available about uterine and embryonic status of ErbB3 or ErbB4 during implantation. We cloned mouse erbB3 and erbB4 cDNAs and examined their expression and bioactivity in the peri-implantation uterus (days 1-8). Two erbB3 (cytoplasmic and extracellular) and three erbB4 (two cytoplasmic and one extracellular) clones were generated. Both forms of the erbB3 clone showed similar transcript profiles, while different transcript profiles were obtained with erbB4 clones. The steady-state levels of erbB3 and erbB4 mRNAs in whole uterine poly(A)+ RNA samples showed little changes during the peri-implantation period, while their unique cell-specific accumulation was noted. erbB3 is predominantly expressed in the epithelial cells, although decidual and embryonic cells also accumulate this mRNA. In contrast, the erbB4 mRNA is primarily expressed in the submyometrial stroma and myometrial connective tissues during this period. Additionally, the extracellular form of the erbB4 clone detected signals in a subpopulation of stromal cells. Autophosphorylation and immunoprecipitation studies provided evidence that uterine ErbB3 and ErbB4 are biologically active. This study provides a comprehensive analysis of possible ligand-receptor signaling schemes for EGF-like ligands in implantation.

Neuregulins signal cells by binding to an activating hetero- and homodimeric forms of the neuregulin receptors HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). Axonally derived neuregulin signals myelin forming cells of the central and peripheral nervous systems through different receptor complexes: oligodendrocytes through erbB2/erbB4 heterodimers and Schwann cells through erbB2/erbB3 heterodimers. Since the leading edge of myelinating cells interacts directly with the axonal surface, we were interested in determining if signaling molecules localized at the leading edge associate with activated neuregulin receptors. We found a novel association between neuregulin receptors and focal adhesion kinase (FAK) in primary cultures of Schwann cells. Following stimulation with ligand, maximal binding of FAK to HER2 occurred by 1 min whereas maximal binding to HER3 was delayed to approximately 7 min. FAK is localized in focal adhesions of Schwann cells. We have previously shown HER2 and HER3 are distributed evenly throughout the plasmalemma. Neuregulins thus use FAK to transmit intracellular signals and the differential kinetics of FAK association with individual neuregulin receptors, as well as its restricted subcellular localization, may play a role in specifying biologic responses.

Neuregulin-1 (NRG1) belongs to a large family of growth and differentiation factors with a key role in the development and maintenance of the brain. Genetic association of NRG1 within brain disorders such as Alzheimer's disease, schizophrenia and neuroprotective properties of certain NRG1 isoforms have led to a variety of studies in corresponding disease models. In the present work, we investigated NRG1 with regard to its peripheral and central biodistribution after systemic application. We first-time radiolabeled the entire biologically active extracellular domain of NRG1 isotype-β1 (NRG1-β1 ECD; aa 2-246) with iodine-125 and administered it peripherally to healthy adult C57Bl6 mice. Blood kinetics and relative organ distribution of (125)I-labeled NRG1-β1 ECD were determined. The blood level of NRG1-β1 ECD peaked within the first hour after intraperitoneal (i.p.) application. The brain-blood ratios of (125)I-labeled NRG1-β1 ECD were time-dependently 150-370% higher compared to the brain impermeable control, (131)I-labeled bovine serum albumin. Autoradiographs of brain slices demonstrated that (125)I-labeled NRG1-β1 ECD accumulated in several regions of the brain e.g. frontal cortex, striatum and ventral midbrain containing the substantia nigra. In addition we found histochemical and biochemical evidence that phosphorylation of the NRG1 prototype receptor ErbB4 was increased in these regions after systemic application of NRG1-β1 ECD. Our data suggest that NRG1-β1 ECD passes the blood-brain barrier and activates cerebral ErbB4 receptors.

Determining molecular mechanisms of human embryo implantation is an extremely challenging task due to the limitation of materials and significant differences underlying this process among mammalian species. Recently, L-selectin and its ligand carbohydrate have been proposed as a system that mediates initial adhesion of human blastocysts to the uterine epithelia. We have also identified trophinin as a unique apical cell adhesion molecule potentially involved in the initial adhesion of trophectoderm of the human blastocyst to endometrial surface epithelia. In the mouse, the binding between ErbB4 on the blastocyst and heparin-binding epidermal growth factor-like growth factor on the endometrial surface enables the initial step of the blastocyst implantation. The evidence suggests that L-selectin and trophinin are included in human embryo implantation. This review summarizes findings relevant to the functions of L-selectin and trophinin in human embryo implantation, and proposes a model that reconciles these cell adhesion mechanisms.

The neuregulin/erbB-signaling axis contributes to the development and growth of multiple mammalian tissues including skeletal muscle. In this study, we sought to characterize the native expression of this system in human skeletal muscle and test the hypothesis that a program of progressive resistance training (PRT) would regulate the expression of neuregulin (NRG) and its cognate receptors. Twelve healthy-male subjects underwent 8-weeks of lower-extremity PRT and muscle biopsies were performed at baseline and following 1- and 8-weeks of the intervention. PRT resulted in significant gains in skeletal muscle strength without appreciable changes in fiber size or myosin heavy chain (MHC) composition. At baseline, Western Blot analysis demonstrated expression of erbB2, erbB3 and erbB4 receptors and multiple NRG isoforms. Following 1- and 8-weeks of PRT, no changes erbB2, erbB4 or NRG expression were observed. ErbB3 expression, however, was significantly increased at both time points compared to baseline. Double labeling of muscle cross-sections revealed increased expression of erbB3 following PRT was not exclusive to fibers staining positive for MHC IIa. Thus, erbB2, erbB3, erbB4 and multiple NRG isoforms are natively expressed in human skeletal muscle. Following PRT, a significant increase in erbB3 was observed. The ability to detect basal expression and alterations in response to physiologic stimuli merit further studies examining the role of this system in skeletal muscle.

Antibodies targeting the extracellular domains of ErbB receptors have been extensively studied for cancer drug development. This work has led to clinical approval of monoclonal antibodies against the well-known oncogenes EGFR and ErbB2. Here we discuss the biological activities of ErbB4, a less-studied member of the EGFR/ErbB growth factor receptor family and speculate on the potential clinical relevance of antibodies targeting ErbB4. In addition to their significance as therapeutics, the role of ErbB4 antibodies in prognostic and predictive applications is surveyed.

Betacellulin (BTC) purified from mouse beta cell tumor (betaTC-3) is a new member of the epidermal growth factor (EGF) family which can bind receptor tyrosine kinase, EGF receptor (erbB1) and erbB4. It has been demonstrated that proBTC mRNA was abundantly expressed in human pancreas tissue, and that BTC converted amylase-secreting rat acinar cell line (AR42J) into insulin-secreting cells, suggesting that BTC might be important for the growth and/or differentiation of islet cells. However, the cell type producing BTC in the pancreas has not been clarified. In this study, we examined the localization of BTC in human pancreas and islet cell tumors. Immunohistochemistry using specific antibodies to human BTC revealed that this protein was produced in alpha cells and duct cells, and probably in beta cells in normal adult pancreas. Furthermore, strong immunoreactivity to BTC was detected in primitive duct cells of the fetal pancreas, and both insulinoma and glucagonoma cells also showed positive immunoreactivity to BTC. EGF receptor (erbB1) and erbB4 were expressed mainly in islet and duct cells, and duct cells, respectively. These results demonstrate the localization of BTC and its receptors, and suggest that BTC may be one of the factors that have physiologically important roles such as growth and differentiation of islet cells in the human pancreas.

Heparin binding EGF-like growth factor (HB-EGF), encoded by the Hegfl gene, is considered as an important mediator of embryo-uterine interactions during implantation in mice. However, it is unknown whether HB-EGF is important for implantation in species with different steroid hormonal requirements. In mice and rats, maternal ovarian estrogen and progesterone (P(4)) are essential to implantation. In contrast, blastocyst implantation can occur in hamsters in the presence of P(4) alone. To ascertain whether HB-EGF plays any role in implantation in hamsters, we examined the expression, regulation and signaling of HB-EGF in the hamster embryo and uterus during the periimplantation period. We demonstrate that both the blastocyst and uterus express HB-EGF during implantation. Hegfl is expressed solely in the uterine luminal epithelium surrounding the blastocyst prior to and during the initiation of implantation. Hypophysectomized P(4)-treated pregnant hamsters also showed a similar pattern of implantation-specific Hegfl expression. These results suggest that uterine Hegfl expression at the implantation site is driven by either signals emanating from the blastocyst or maternal P(4), but not by maternal estrogen. However, in ovariectomized hamsters, uterine induction of Hegfl requires the presence of estrogen and activation of its nuclear receptor (ER), but not P(4). This observation suggests an intriguing possibility that an estrogenic or unidentified signal from the blastocyst is the trigger for uterine HB-EGF expression. An auto-induction of Hegfl in the uterus by blastocyst-derived HB-EGF is also a possibility. We further observed that HB-EGF induces autophosphorylation of ErbB1 and ErbB4 in the uterus and blastocyst. Taken together, we propose that HB-EGF production and signaling by the blastocyst and uterus orchestrate the 'two-way' molecular signaling to initiate the process of implantation in hamsters.

Glioblastoma multiforme is the most common type of primary brain tumor in adults. WWOX is a tumor suppressor gene involved in carcinogenesis and cancer progression in many different neoplasms. Reduced WWOX expression is associated with more aggressive phenotype and poor patient outcome in several cancers. We investigated alternations of WWOX expression and its correlation with proliferation, apoptosis and signal trafficking in 67 glioblastoma multiforme specimens. Moreover, we examined the level of WWOX LOH and methylation status in WWOX promoter region. Our results suggest that loss of heterozygosity (relatively frequent in glioblastoma multiforme) along with promoter methylation may decrease the expression of this tumor suppressor gene. Our experiment revealed positive correlations between WWOX and Bcl2 and between WWOX and Ki67. We also confirmed that WWOX is positively correlated with ErbB4 signaling pathway in glioblastoma multiforme.

The class I tyrosine kinase growth-factor receptors include epidermal growth factor receptor (EGFR), ErbB2 (c-erbB-2, HER-2/neu), ErbB3 and ErbB4. To elucidate their role in the regulation of homeostasis and carcinogenesis, we examined the expression of the receptors in normal urothelium and in urothelial carcinoma by immunohistochemistry. EGFR was expressed in the basal cells of normal urothelium, while ErbB2, ErbB3 and ErbB4 were present mainly in the superficial layer. A distinct reciprocal distribution was observed between the EGFR and the remaining members of the subclass (P = 0.0001). Both BCL-2 protein and Ki-67 antigen (MIB-1) showed a strong positive association with EGFR (P = 0.002) and an inverse correlation with ErbB2, ErbB3 or ErbB4 (P = 0.0004, 0.0000, and 0.001, respectively). With regard to carcinoma, there was no important relationship between receptor overexpression and tumour grading (P>> 0.1), while only EGFR overexpression was correlated with muscular invasion (P = 0.02). Coexpression of EGFR-ErbB3 and ErbB3-ErbB4 was more often detected in high-grade tumours and correlated with the extent of tumour invasion. Our data indicate that class I receptors are differentially expressed in normal urothelium in vivo, but an orchestrated expression pattern does not exist during tumorigenesis.

Schizophrenia is a chronic, disabling neuropsychiatric disorder with complex genetic origins. The development of strategies for genome manipulation in rodents provides a platform for understanding the pathogenic role of genes and for testing novel therapeutic agents. Neuregulin 1 (NRG1), a critical developmental neurotrophin, is associated with schizophrenia. The NRG1 gene undergoes extensive alternative splicing and, to date, little is known about the neurobiology of a novel NRG1 isoform, NRG1-IV, which is increased in the brains of individuals with schizophrenia and associated with genetic risk variation. Here, we developed a transgenic mouse model (NRG1-IV/NSE-tTA) in which human NRG1-IV is selectively overexpressed in a neuronal specific manner. Using a combination of molecular, biochemical, electrophysiological, and behavioral analyses, we demonstrate that NRG1-IV/NSE-tTA mice exhibit abnormal behaviors relevant to schizophrenia, including impaired sensorimotor gating, discrimination memory, and social behaviors. These neurobehavioral phenotypes are accompanied by increases in cortical expression of the NRG1 receptor, ErbB4 and the downstream signaling target, PIK3-p110δ, along with disrupted dendritic development, synaptic pathology, and altered prefrontal cortical excitatory-inhibitory balance. Pharmacological inhibition of p110δ reversed sensorimotor gating and cognitive deficits. These data demonstrate a novel role for NRG1-IV in learning, memory, and neural circuit formation and a potential neurobiological mechanism for schizophrenia risk; show that deficits are pharmacologically reversible in adulthood; and further highlight p110δ as a target for antipsychotic drug development.

Schizophrenia is a disabling psychiatric disorder with neurodevelopmental origins. Genes that increase risk for schizophrenia have been identified. Understanding how these genes affect brain development and function is necessary. This work is the first report of a newly generated humanized transgenic mouse model engineered to express human NRG1-IV, an isoform of the NRG1 (Neuregulin 1) gene that is increased in the brains of patients with schizophrenia in association with genetic risk. Using behavioral neuroscience, molecular biology, electrophysiology, and pharmacology, we identify a role for NRG1-IV in learning, memory, and cognition and determine that this relates to brain excitatory-inhibitory balance and changes in ErbB4/PI3K/AKT signaling. Moreover, the study further highlights the potential of targeting the PI3K pathway for the treatment of schizophrenia.

It is increasingly recognized that drug-resistant cells undergo transitions not directly linked to "classical" drug resistance. We examined the expression of growth factors, growth factor receptors, and the estrogen receptor in 17 drug-resistant and 2 revertant human breast cancer sublines to provide an understanding of the phenotypic changes that occur and how these changes could affect the biology of the cell. These sublines were derived from five parental human breast cancer cell lines (MCF-7, ZR75B, T47D, MDA-MB-231, and MDA-MB-453). The expression of estrogen receptor was absent or decreased in 6 of the 15 resistant MCF-7, ZR75B, and T47D sublines. Increases of as much as 49-fold compared to parental levels were observed in transforming growth factor alpha, epidermal growth factor receptor, c-erbB2, and/or c-erbB3 mRNA expression in 14 of the 17 resistant sublines. Altered amphiregulin and insulin-like growth factor-I receptor expression was observed in nine and four drug-resistant sublines, respectively. No major alterations were observed in epidermal growth factor and c-erbB4 expression. Few alterations were observed in two sublines derived from estrogen receptor-negative cells. Higher levels of phosphotyrosine residues were detected in a subset of the resistant sublines, indicating an increased tyrosine kinase activity in these cells. Interestingly, decreased growth rates were observed in all of the sublines, despite up-regulated growth factor-related gene expression. Taken together, these data suggest that loss of estrogen receptor, increased expression of growth factor pathway genes, and decreased growth rate regularly occur in drug-resistant breast cancer cells. Although we do not know whether the altered expression of growth factor pathway genes is linked as a cause or a consequence of the reduced growth rate, it is well established that decreased growth rate confers drug resistance. These phenotypic changes in drug-resistant human breast cancer cells could serve to initiate, support, or extend the drug resistance.

The ErbB receptor tyrosine kinase family consists of the epidermal growth factor (EGF) receptor (ErbB1) and three related receptors (ErbB2, ErbB3, ErbB4). Their intrinsic tyrosine kinases can be activated by receptor-dimerization induced by numerous ligands or overexpression. ErbB receptors are frequently overexpressed in breast cancer, and their overexpression is associated with protection from apoptosis. To directly assess their role in apoptosis sensitivity of breast cancer cells, we established MCF-7 breast carcinoma cell lines overexpressing each ErbB receptor alone or in all possible pairs. Overexpression of ErbB1, ErbB2 and ErbB4 receptors was enough to activate them as judged by their phosphorylation, whereas co-expression of other ErbB receptors was necessary for the phosphorylation of the ErbB3. Surprisingly, overexpression of the ErbB receptors even when combined with treatment with their ligands (EGF, transforming growth factor alpha, betacellulin, neuregulins) failed to protect the MCF-7 cells from cell death induced by either tumor necrosis factor (TNF) or serum starvation. During starvation TGF-alpha, however, increased the cell size of the ErbB1 overexpressing cell line, and neuregulin1-beta1 increased that of all cell lines. In conclusion, our data does not support the role of ErbB receptors in the regulation of cell death induced by TNF or serum starvation, and the observed association in breast cancer may be due to other concomitant changes.

The ErbB family of receptor tyrosine kinases comprises four members: epidermal growth factor receptor (EGFR)/ErbB1, HER2/ErbB2, ErbB3 and ErbB4, and plays roles in signal transduction at the plasma membrane upon ligand stimulation. Stimulation with neuregulin-1 (NRG-1) cleaves ErbB4 and releases the ErbB4 intracellular domain (4ICD) that translocates into the nucleus to control gene expression. However, little is known about the regulation of 4ICD nuclear signaling through tyrosine phosphorylation. We show here that 4ICD nuclear signaling is antagonized by EGF-induced c-Src activation through EGFR. Generation of 4ICD by NRG-1 leads to increased levels of trimethylated histone H3 on lysine 9 (H3K9me3) in a manner dependent on the nuclear accumulation of 4ICD and its tyrosine kinase activity. Once EGF activates c-Src downstream of EGFR concomitantly with NRG-1-induced ErbB4 activation, c-Src associates with phospho-Tyr950 and phospho-Tyr1056 on 4ICD, thereby decreasing nuclear accumulation of 4ICD and inhibiting an increase of H3K9me3 levels. Moreover, 4ICD-induced transcriptional repression of the human telomerase reverse transcriptase (hTERT) is inhibited by EGF-EGFR-Src signaling. Thus, our findings reveal c-Src-mediated inhibitory regulation of ErbB4 nuclear signaling upon EGFR activation.

The neu (c-erbB-2, HER2) proto-oncogene encodes a receptor tyrosine kinase that is a member of an important growth factor receptor family which includes the epidermal growth factor receptor (EGFR, ErbB1), ErbB3 and ErbB4. The neu is found over-expressed in 20-30% of human breast tumors. The c-erbB-2 is sufficient for the induction of mammary tumorigenesis in transgenic mice and the pathology of these mammary tumors strongly resembles human breast cancer. Murine transgenic models engineered to recapitulate human breast cancer provide an excellent and straightforward approach to dissect the molecular mechanisms governing the onset and progression of this disease. The molecular mechanisms by which ErbB-2 transforms cells involves direct effects on components of the cell-cycle regulatory apparatus. Recent studies have demonstrated a key role for components of the cell-cycle, in particular cyclin D1 and p27Kip1 (p27) in the onset and progression of ErbB-2-induced murine mammary tumorigenesis. Such studies have provided further impetus to therapeutics targeting these cell-cycle proteins.

The ErbB4 receptor tyrosine kinase is expressed at high levels in human and mouse colitis, and inhibits colon epithelial cell apoptosis in the presence of proinflammatory cytokines. In this study, we investigated the molecular mechanisms responsible for ErbB4-induced cell survival. In cultured mouse colon epithelial cells, ErbB4 overexpression resulted in increased levels of cyclooxygenase-2 (COX-2) mRNA and protein; in contrast, ErbB4 knockdown with siRNA blocked COX-2 accumulation in response to tumor necrosis factor. Although ErbB4 is expressed as up to four isoforms in epithelial tissues, its ability to promote COX-2 expression was isoform independent. ErbB4-stimulated COX-2 induction was associated with an increase in mRNA half-life and was blocked by inhibition of Src, phosphatidylinositol (PI) 3-kinase, or epidermal growth factor receptor (EGFR). Furthermore, ErbB4 expression promoted EGFR phosphorylation in the presence of heregulin, implicating ErbB4-EGFR heterodimerization in these responses. As to the cellular responses to ErbB4 activation, increased survival of ErbB4-expressing cells in the presence of proinflammatory cytokines was sensitive to the COX-2 inhibitor celecoxib. Furthermore, ErbB4-overexpressing cells acquired the ability to form colonies in soft agar, indicative of cellular transformation, also in a celecoxib-sensitive manner. Together our data indicate that ErbB4 is a key regulator of COX-2 expression and cellular survival in colon epithelial cells, acting in concert with EGFR through a Src- and PI 3-kinase-dependent mechanism. These results suggest that chronic overexpression of ErbB4 in the context of inflammation could contribute to colitis-associated tumorigenesis by inhibiting colonocyte apoptosis.

We previously found that EGF (epidermal growth factor) increases the EGFR (EGF receptor) kinase-binding affinity towards the major tyrosine phosphorylation sites in downstream adaptor proteins such as Gab1 (Grb2-associated binding protein 1) and Shc [Src homology 2 (SH2) domain and collagen containing protein], but not that towards EGFR autophosphorylation sites [Fan, Wong, Deb and Johnson (2004) J. Biol. Chem. 279 , 38143-38150]. EGFR activation can also result in transphosphorylation of tyrosine resides in the C-terminal region of the related receptors ErbB2, ErbB3 and ErbB4 in heterodimers which are formed upon ligand stimulation. In the present study, we investigated the specificity of EGFR kinase by comparing the steady state kinetic parameters for peptides derived from all four ErbBs in the absence or presence of EGF. Our results demonstrated that (i) EGFR kinase can efficiently phosphorylate a broad range of diverse peptide sequences representing ErbB sites; (ii) certain ErbB2, ErbB3 and ErbB4 sites had higher specificity constants than any EGFR sequence and (iii) EGF stimulation consistently increases the k(cat) approx. 5-fold, but does not significantly alter the K(m) for any ErbB peptides. Furthermore, peptides containing lysine at position -2 or -3 N-terminal to the target tyrosine were found to be poor EGFR kinase substrates, and substitution of these lysines with glutamine decreased the K(m) and increased the k(cat) for these substrates. We conclude that EGFR kinase-mediated ErbB transphosphorylations are mostly controlled at the level of oligomerization, and not by a preference of the EGFR kinase for phosphorylation sites in any particular ErbB. The results also demonstrated that, unlike phosphorylation sites in select downstream targets, EGF does not regulate the recognition of phosphorylation sites in the C-terminal region of any of the ErbBs.

Neuregulin-1 (NRG-1), a cardioactive growth factor released from endothelial cells, is indispensable for cardiac development, structural maintenance, and functional integrity of the heart. In recent years, a growing number of studies have focused on NRG-1 and members of the ErbB family that serve as receptors for NRG-1 in order to better understand the role of this signaling pathway in physiology and pathophysiology of the heart. An essential role for NRG-1 and ErbB in heart development and functionality has been suggested by studies in conditional NRG-1/ErbB-deficient mice and by the cardiac-related side effects of anti-ErbB2 antibody therapies used for treatment of breast cancer. In vitro and in vivo studies using recombinant human neuregulin-1 (rhNRG-1), which contains the epidermal growth factor (EGF)-like domain (necessary for ErbB2/ErbB4 activation), have further supported the hypothesis that NRG-1 plays an important role in heart function. Consistent with other studies, expression of rhNRG-1 not only restored normal cardiomyocytic structure altered by nutritional deficiency in cell cultures, but also improved the pumping function of the heart in several animal models of chronic heart failure (CHF). As a result of these findings, proteins involved in the NRG-1/ErbB-signaling pathway have been explored as potential drug targets for treatment of heart failure. Clinical trials to evaluate the safety and efficacy of rhNRG-1 have been conducted in both China and Australia. As predicted, rhNRG-1 treatment improved both cardiac function and reversed remodeling of the heart. Therefore, rhNRG-1 may represent a new drug for treatment of CHF with a novel therapeutic mechanism.

Although expression of the ErbB4 receptor tyrosine kinase in breast cancer is generally regarded as a marker for favorable patient prognosis, controversial exceptions have been reported. Alternative splicing of ErbB4 pre-mRNAs results in the expression of distinct receptor isoforms with differential susceptibility to enzymatic cleavage and different downstream signaling protein recruitment potential that could affect tumor progression in different ways. ErbB4 protein expression from nontransfected cells is generally low compared with ErbB1 in most cell lines, and much of our knowledge of the role of ErbB4 in breast cancer is derived from the ectopic overexpression of the receptor in non-breast-derived cell lines. One of the primary functions of ErbB4 in vivo is in the maturation of mammary glands during pregnancy and lactation induction. Pregnancy and extended lactation durations have been correlated with reduced risk of breast cancer, and the role of ErbB4 in tumor suppression may therefore be linked with its role in lactation. Most reports are consistent with a role for ErbB4 in reversing growth stimuli triggered by other ErbB family members during puberty. In this report, we provide a systems-level examination of several reports highlighting the seemingly opposing roles of ErbB4 in breast cancer and potential explanations for the discrepancies and draw the conclusion that future studies examining the function of ErbB4 in breast cancer should also take into account the pregnancy history, lactation status, and hormone supplementation or ablation history of the patient from whom the tumor or tumor cells are derived.

The beta isoform of Neuregulin-1 (NRG-1β), along with its receptors (ErbB2-4), is required for cardiac development. NRG-1β, as well as the ErbB2 and ErbB4 receptors, is also essential for maintenance of adult heart function. These observations have led to its evaluation as a therapeutic for heart failure. Animal studies and ongoing clinical trials have demonstrated beneficial effects of two forms of recombinant NRG-1β on cardiac function. In addition to the possible role for recombinant NRG-1βs as heart failure therapies, endogenous NRG-1β/ErbB signaling appears to play a role in restoring cardiac function after injury. The potential mechanisms by which NRG-1β may act as both a therapy and a mediator of reverse remodeling remain incompletely understood. In addition to direct effects on cardiac myocytes NRG-1β acts on the vasculature, interstitium, cardiac fibroblasts, and hematopoietic and immune cells, which, collectively, may contribute to NRG-1β's role in maintaining cardiac structure and function, as well as mediating reverse remodeling.

Cardiotoxicity is a common and potentially devastating side effect of antineoplastic drug therapy. This empiric observation is seen as paradoxical given that the cardiomyocyte is considered to be a terminally differentiated cell. Despite the fact that these cells do not divide after birth, adult cardiomyocytes may become "innocent bystander" targets of anticancer drugs designed to interfere with cell signaling pathways in rapidly proliferating cells. In breast cancer clinical trials, treatment with the erbB2 receptor antibody trastuzumab combined with anthracyclines has been associated with an increased risk for the development of cardiac pump failure. Trastuzumab/anthracycline cardiomyopathy may be the first clinically significant cardiotoxicity to emerge from signal transduction therapeutics. The erbB2 receptor tyrosine kinase is known to have a critical role in cardiac development. In addition, erbB2 is thought to participate in an important pathway for growth, repair, and survival of adult cardiomyocytes as part of a signaling network that involves neuregulins and the neuregulin receptor erbB4. However, erbB2 levels in the adult heart are low when compared with the levels found in erbB2-overexpressing breast cancer cells that are the intended targets of trastuzumab therapy. Thus, trastuzumab-associated cardiotoxicity must be explained by some alternative mechanism. After confirming that trastuzumab is capable of inducing tyrosine phosphorylation of the human cardiomyocyte erbB2 protein, a novel system for culturing human myocardium was developed in our laboratory. We used this system to study the effects of trastuzumab on human cardiomyocytes in vitro and observed trastuzumab-induced structural and functional changes in human cardiomyocytes that were at least partially reversible with the addition of recombinant neuregulins. The results obtained in these experiments support a direct action of trastuzumab on human cardiomyocytes. In addition, these data provide insight regarding potential molecular mechanisms. Most importantly, these data draw attention to the inherent risk of cardiotoxicity associated with a newly emerging class of antineoplastic drugs that interfere with signal transduction pathways.

OBJECTIVE

The receptor tyrosine kinase ERBB4/HER4 plays a role in cell division, migration, differentiation, as well as apoptosis, and is frequently overexpressed in breast and colorectal tumors. To understand the role of genetic variations in the regulation of ERBB4 expression, we identified new polymorphisms and investigated their functional implication and risk association with breast and colorectal cancer.

METHODS

We screened colorectal tumors from 92 patients for genetic variants at the ERBB4 ATG -1000 bp 5'-regulatory region by denaturing high-performance liquid chromatography and sequencing. Variants were subjected to DNA-protein interaction analyses (electrophoretic mobility shift assay), reporter gene assays in breast cancer cell lines MDA134 and MDA157, and immunohistochemical analyses of breast tumors. We established genotype frequencies within a breast cancer case-control collection (1,021 cases, 1,015 population-based controls) and a colorectal cancer case-control collection (459 cases, 569 blood donors) using matrix-assisted laser desorption ionization/time of flight mass spectrometry. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were assessed by multivariate logistic regression.

RESULTS

We identified five new germ line variants -815 A>T, -782 G>T, -638 insTC, -267 C>G, and -219 del10bp. Two variants showed in vitro functional effects. The -782T allele showed lower protein binding affinity and lower promoter activity compared with the -782G allele, however, the -815T allele showed higher protein binding affinity and higher promoter activity. The -782T variant was identified as a risk allele for breast and colorectal cancer (OR, 1.59; 95% CI, 1.06-2.34 and OR, 2.21; 95% CI, 1.22-3.99, respectively).

CONCLUSIONS

The ERBB4 -782 G>T polymorphism, by virtue of its in vitro functional implication and incidence, is a risk factor for breast and colorectal cancer.

Ligand-receptor signaling with the epidermal growth factor (EGF) family of growth factors in the uterus and embryo is considered to be important for implantation. The EGF family includes EGF, transforming growth factor-alpha, heparin binding-EGF, amphiregulin, beta-cellulin, epiregulin, and heregulins, whereas the receptor family (the erbB genes) consists of erbB1 (EGF-receptor, EGF-R), erbB2, erbB3, and erbB4. Interactions of uterine EGF-R with EGF-like ligands have been examined, but limited information is available regarding the status of other receptor subtypes. Thus, we examined the expression of the erbB2 gene in the mouse uterus during the periimplantation period (days 1-8 of pregnancy) and after 17 beta-estradiol and/or progesterone stimulation. Northern blot hybridization detected two transcripts (approximately 4.0 and 5.0 kb) of erbB2 messenger RNA (mRNA) in day 1-8 uterine polyadenylated RNA samples. In situ hybridization experiments showed unique uterine cell-specific erbB2 mRNA distribution. On days 1-4, unlike the full-length erbB1 mRNA which is not expressed in the uterine epithelium, the erbB2 mRNA was detected primarily in epithelial cells; the day 1 uterus showed the highest accumulation. On day 5, the epithelium and the decidualizing stromal cells around the implanting blastocyst exhibited accumulation of this mRNA. On days 6-8, the accumulation persisted in the epithelium at both the implantation and interimplantation sites in addition to modest levels of signals in the secondary decidual zone. On days 7 and 8, accumulation of the erbB2 mRNA was also prominent in the trophoblastic giant cells. Western blotting detected a predicted protein of 185 kDa in day 4 uterine membrane preparations. Results of immunocytochemistry demonstrated colocalization of the erbB2 protein with its mRNA in the periimplantation uterus. The uterine ErbB2 underwent phosphorylation by several members of the EGF family. Treatment of adult ovariectomized mice with 17 beta-estradiol, but not progesterone, up-regulated the expression of the erbB2 mRNA by more than 3.5-fold, as determined by quantitative reverse transcription-PCR, and this increase was limited to the epithelium, as revealed by in situ hybridization. Collectively, the results place ErbB2 as a potential candidate receptor subtype for interaction with the EGF-related ligands in epithelial cell proliferation/differentiation during the preimplantation period and stromal cell proliferation/decidualization during the postimplantation period.