ErbB Ligands Research Articles

Overexpression of CD44 in acquired endocrine resistant breast cancer modulates erbB activity and promotes an invasive phenotype.

Abstract Abstract #3027 Background: Acquired endocrine resistance in breast cancer cells is accompanied by altered growth factor receptor signalling and a highly migratory cell phenotype. In tamoxifen-resistant ('TamR') MCF7 cells, our microarray analysis has demonstrated elevated levels of CD44, a transmembrane glycoprotein, known to interact with and modulate the function of a number of growth factor receptors and plays a major role in tumour metastasis. Here, we have explored the role of CD44 as a modulator of erbB family activity in TamR cells and evaluated the expression of CD44 in a series of breast cancer patients on endocrine therapy.
 Methods: RT-PCR was performed to confirm overexpression of CD44 at the gene level in TamR cells. Western blotting and immunocytochemistry were used to investigate expression of CD44 at the protein level together with the activity of erbB signalling pathways following stimulation of CD44 by its ligand, hyaluronan (HA), or following stimulation with erbB ligands (TGFα, heregulin β1) after knockdown of CD44 expression using siRNA. Immunofluorescence microscopy was used to determine association between CD44 and erbB family members. Cell migration was assessed by seeding cells onto fibronectin-coated microporous inserts for 24 hours. CD44 expression levels were evaluated using paraffin-embedded tissue sections in a series of primary breast cancer tissues (n=103) from patients receiving endocrine therapy by immunohistochemical staining.
 Results: CD44 was overexpressed at the gene and protein level in TamR cells versus their endocrine sensitive counterparts. siRNA-mediated suppression of CD44 expression reduced basal EGFR/Her2 activity in TamR cells together with suppressing their migratory phenotype. siRNA-mediated inhibition of CD44 expression greatly attenuated the ability of erbB ligands to activate erbB receptors and inhibited TGFα and heregulin β1-induced migration. Conversely, stimulation of TamR, but not MCF7 cells, with HA promoted activation of erbB family members together with their downstream signalling intermediates ERK1/2, Src and AKT. Analysis of CD44 expression in clinical tissue revealed an association between elevated CD44 expression and a shortened response to endocrine therapy.
 Conclusions: These data demonstrate that overexpression of CD44 accompanies endocrine resistance where it enhances the sensitivity of these cells to factors (erbB ligands, HA) found within the tumour microenvironment. This may implicate CD44 in loss of endocrine response clinically and, therefore, provide a novel therapeutic target in endocrine resistant breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3027.

Transforming growth factor beta engages TACE/ADAM17 and ErbB3 to activate PI3K/Akt in HER2-overexpressing breast cancer and desensitizes cells to trastuzumab.

Abstract Abstract #35 TGFb signaling synergizes with HER2 in breast cancer progression. Exogenous TGFb or expression of an activated TGFb type I receptor (Alk5T204D) in MCF10A/HER2 and BT474 cells activated PI3K/Akt and enhanced survival and migration. In both cell types, TGFb stimulated P-EGFR and P-HER2 as well as the association of p85, the regulatory subunit of PI3K, with Y1289 P-ErbB3 which, in turn, activated PI3K/Akt. RNA interference of ErbB3 or treatment with pertuzumab, an antibody that blocks HER2-mediated activation of ErbB3, blocked TGFb-induced P-ErbB3, P-Akt, and cell motility. Treatment with TGFb or expression of Alk5TD increased protein levels of secreted TGFalpha, amphiregulin, and heregulin without a change in mRNA levels. The increase on secreted ErbB ligands without a change in mRNA levels suggested increased shedding of ErbB pro-ligands by tumor necrosis factor a-converting enzyme (TACE). Transfection of siRNA against human TACE but not control siRNA abrogated TGFb-stimulated ErbB receptor phosphorylation, P-Akt, and cell invasiveness. Transfection of full-length mouse TACE but not truncated TACE lacking the cytoplasmic domain reconstituted TGFb-induced ErbB phosphorylation in cells transfected with human TACE siRNA. TGF-b increased TACE phosphorylation in both serine and threonine as measured by P-Ser and P-Thr immunoblots of TACE pull downs. Incubation of TACE with Alk5TD in the presence of ATP in vitro resulted in both Ser and Thr phosphorylation in full-length but not truncated TACE. Further, addition of TGFb or expression of Alk5TD desensitized SKBR3 and BT474 cells to trastuzumab. Treatment with the type I TGFb receptor (Alk5) small molecule inhibitor LY2109761, the PI3K inhibitor LY294002, or siRNA against ErbB3 or TACE restored the inhibitory effect of trastuzumab. Finally, we generated an active Alk5 expression signature by selecting genes that were differentially expressed between BT474/Alk5TD and BT474/vector cells. Mapping of this signature to a previously published 295-breast tumor array (van de Vijver et al. and Chang et al.) revealed 90 of 271 genes in the Alk5TD signature with a >1.5-fold change from the median expression which correlated with HER2-positive and basal-like tumors and conferred a worse recurrence-free (RFS) and overall survival (OS) (RFS p=0.05, OS p=0.01). Hierarchical clustering analysis to an array data set reported by Harris et al. in 22 patients with HER2+ breast cancer treated with neoadjuvant trastuzumab and vinorelbine (Clin. Cancer Res. 2007) showed that the Alk5TD signature correlated with tumors that did not respond clinically. These data suggest that TGFb potentiates signaling downstream ErbB receptors via TACE, ErbB3, and PI3K, thus contributing to tumor progression and resistance to anti-HER2 therapies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 35.

ErbB receptors protect the perfused heart against injury induced by epinephrine combined with low-flow ischemia

ErbB receptor tyrosine kinases are important in maintaining the long-term structural integrity of the heart and in the induction of hypertrophy. In addition, in vivo activation of ErbB1 by epidermal growth factor (EGF) protects the heart against acute stress-induced damage. We examined here whether the ErbB sytem acutely protects the isolated heart in which stress was induced in vitro by ischemia combined with epinephrine infusion (EPI). In perfused mouse hearts, EGF induced Tyr-phosphorylation of ErbB1 but not ErbB2. Neuregulin-1β (NRG-1β) induced Tyr-phosphorylation of both ErbB4 and ErbB2. We also found differences in the signaling cascades activated by each growth factor. To stress the perfused mouse heart, we combined EPI with low-flow ischemia. This resulted in (i) loss of left ventricle contraction force ( + dP/dtmax) and developed pressure (LVDP) after a short period of hypercontractility, (ii) enhanced anaerobic metabolism (lactate production), and (iii) myocyte injury (lactate dehydrogenase (LDH) release). EGF and NRG-1β had different effects on stressed-heart contractility. EGF reduced to a half the loss of both + dP/dtmax and LVDP. In contrast, NRG-1β exacerbated the hypercontractility soon after reperfusion. This is coincident with a transient increase in coronary flow after reperfusion. In spite of these differences in contraction, both EGF and NRG-1β induced similar early protection as shown by the reduction of LDH release. Our results show that the ErbB system protects the perfused heart against damage induced by acute stress. They reinforce the relevance of ErbB receptors and ligands in cardiac physiology.

Gene Expression Profiling of ErbB Receptors and Ligands in Human Meningiomas

ErbB family receptors mediate major cellular functions implied in tumorigenesis, though their role in meningiomas was not thoroughly studied. Meningiomas represent 30% of primary cranial tumors, are mostly benign, and prevail in the second half of life. Tumor therapy requires information about molecular alterations, thus we studied expression of ErbB receptor and ligand genes by real-time RT-PCR in different meningioma grades. Receptors were overexpressed (ErbB1, ErbB2) or underexpressed (ErbB3, ErbB4). Ligands EGF, TGFA, AREG, DTR, BTD were underexpressed and the neuregulins were overexpressed or underexpressed. A strong ErbB1–ErbB2 correlation was found. These data might be useful for gene therapy.

Activation of epidermal growth factor receptor ErbB1 attenuates inhibitory synaptic development in mouse dentate gyrus

Ligands for the epidermal growth factor receptor ErbB1, such as epidermal growth factor (EGF) and transforming growth factor α (TGFα), negatively regulate synaptic maturation of GABAergic neurons in the developing neocortex. Here, we evaluated the effects of these ligands in vivo on developing inhibitory neurons in the dentate gyrus. Hippocampal slices were prepared from postnatal mice repeatedly challenged with EGF or from transgenic mice overexpressing TGFα. We monitored paired pulse depression of field population spikes evoked by perforant path stimulation to estimate the strength of local inhibition. Administration of EGF increased the paired pulse ratio, suggesting a reduction of inhibitory strength. A similar reduction was observed in TGFα transgenic mice. Monitoring miniature and evoked synaptic currents, we estimated EGF effects on synaptic input and output of GABAergic neurons. EGF treatment diminished the amplitude of excitatory postsynaptic currents (EPSCs) in the GABAergic neurons without affecting their miniature EPSCs. EGF also affected output strength of the GABAergic neurons: The frequency of miniature inhibitory postsynaptic currents (IPSCs) and the evoked IPSC/evoked EPSC ratio were decreased in granule cells. In parallel, EGF down-regulated the protein level of vesicular GABA transporter. Thus, ErbB1 ligands influence GABAergic inhibitory synaptic transmission in the developing dentate gyrus.

Ventricular ErbB2/ErbB4 activation and downstream signaling in pacing-induced heart failure

The neuregulin-1 (NRG-1)/ErbB system has emerged as a cardioprotective system that becomes activated during myocardial stress, most convincingly shown in response to cardiotoxic chemotherapy. Direct evidence of increased ventricular ErbB receptor activity in heart failure unrelated to cardiotoxic drugs is, however, limited. We investigated changes in NRG-1 expression, ErbB receptor phosphorylation and downstream activation of intracellular ErbB targets during rapid pacing and progressive ventricular dysfunction in the dog. Heart failure was induced in dogs by 7 weeks of rapid pacing. Ventricular function was assessed by echocardiography. Messenger RNA expression was investigated in ventricular biopsies using quantitative PCR. Activation of NRG-1/ErbB signaling and of downstream targets was investigated using immunoprecipitation and/or Western blotting. Over the course of 7 weeks of pacing and ventricular dilatation, ventricular levels of NRG-1, but not of other ErbB4 ligands, and of ADAM19, a protease promoting NRG-1 release, progressively increased. In parallel, levels of activated ErbB2 and ErbB4, phosphorylated at tyrosine residues 877/1248 and 1284 respectively, became progressively higher. Similarly, levels of total and phosphorylated PI-3 kinase increased. Surprisingly, however, and in contrast with activation of downstream targets of ErbB receptors in normal hearts, Akt and ERK1/2, remained inactivated. This study shows that ventricular ErbB2 and ErbB4 receptors become activated during the development of pacing-induced heart failure, but that downstream signaling is, at least partly, abrogated. Abrogation of cardioprotective signaling after ErbB activation is an unanticipated phenomenon in the progression of heart failure with possibly major pathophysiological significance. The underlying mechanisms should be further elucidated.

EBP1, an ErbB3-binding protein, is decreased in prostate cancer and implicated in hormone resistance

Aberrant activation of the androgen receptor (AR) by the ErbB2/ErbB3 heterodimer contributes to the development of hormone resistance in prostate cancer. EBP1, an ErbB3-binding protein, acts as an AR corepressor. As EBP1 is decreased in preclinical models of hormone-refractory prostate cancer, we studied the expression of EBP1 in human prostate cancer. We found that the expression of the EBP1 gene was significantly decreased in prostate cancer tissues compared with benign prostate at both mRNA and protein levels. Restoration of EBP1 expression in the hormone-refractory LNCaP C81 cell line led to an amelioration of the androgen-independent phenotype based on established biological criteria and a reduction in the expression of a cohort of AR target genes. The ability of the ErbB3 ligand heregulin (HRG) to stimulate growth and AKT phosphorylation of hormone-refractory prostate cancer cells was abolished. Abrogation of EBP1 expression by short hairpin RNA in hormone-dependent LNCaP cells, which undergo apoptosis in response to HRG, resulted in HRG-stimulated cell growth. Restoration of EBP1 expression decreased the tumorigenicity of C81 xenografts in female mice, whereas elimination of EBP1 expression enhanced the ability of LNCaP cells to grow in female mice. Our data support a role for EBP1 in the development of hormone-refractory prostate cancer via inhibition of both AR- and HRG-stimulated growth and present a novel strategy for treating androgen-refractory prostate cancer.

Differential erbB signaling in astrocytes from the cerebral cortex and the hypothalamus of the human brain

Studies in rodents have shown that astroglial erbB tyrosine kinase receptors are key regulatory elements in neuron-glia communication. Although both astrocytes and deregulation of erbB functions have been implicated in the pathogenesis of many common human brain disorders, erbB signaling in native human brain astrocytes has never been explored. Taking advantage of our ability to perform primary cultures from the cortex and the hypothalamus of human fetuses, we conducted a thorough analysis of erbB signaling in human astrocytes. We showed that human cortical astrocytes express erbB1, erbB2, and erbB3, whereas human hypothalamic astrocytes express erbB1, erbB2, and erbB4 receptors. Ligand-dependent activation of different erbB receptor heterodimeric complexes in these two populations of astrocytes translated into different morphological and proliferative responses. Although morphological plasticity was more pronounced in hypothalamic astrocytes than in cortical astrocytes, the former showed a lower mitogenic potential. Decreasing erbB4 expression via siRNA-mediated gene knockdown revealed that erbB4 constitutively restrains basal proliferative activity in hypothalamic astrocytes. We further show that treatment of human astrocytes with a protein kinase C activator results in rapid tyrosine phosphorylation of erbB receptors that involves cleavage of endogenous membrane bound erbB ligands by metalloproteinases. Together, these results indicate that erbB signaling in primary human brain astrocytes is functional, region-specific, and can be activated in a paracrine and/or autocrine manner. In addition, by revealing that some aspects of astroglial erbB signaling are different between human and rodents, our results provide a molecular framework to explore the potential involvement of astroglial erbB signaling deregulation in human brain disorders.

Transforming Growth Factor β Engages TACE and ErbB3 To Activate Phosphatidylinositol-3 Kinase/Akt in ErbB2-Overexpressing Breast Cancer and Desensitizes Cells to Trastuzumab

In HER2-overexpressing mammary epithelial cells, transforming growth factor beta (TGF-beta) activated phosphatidylinositol-3 kinase (PI3K)/Akt and enhanced survival and migration. Treatment with TGF-beta or expression of an activated TGF-beta type I receptor (Alk5 with the mutation T204D [Alk5(T204D)]) induced phosphorylation of TACE/ADAM17 and its translocation to the cell surface, resulting in increased secretion of TGF-alpha, amphiregulin, and heregulin. In turn, these ligands enhanced the association of p85 with ErbB3 and activated PI3K/Akt. RNA interference of TACE or ErbB3 prevented TGF-beta-induced activation of Akt and cell invasiveness. Treatment with TGF-beta or expression of Alk5(T204D) in HER2-overexpressing cells reduced their sensitivity to the HER2 antibody trastuzumab. Inhibition of Alk5, PI3K, TACE, or ErbB3 restored sensitivity to trastuzumab. A gene signature induced by Alk5(T204D) expression correlated with poor clinical outcomes in patients with invasive breast cancer. These results suggest that by acting on ErbB ligand shedding, an excess of TGF-beta may result in (i) conditioning of the tumor microenvironment with growth factors that can engage adjacent stromal and endothelial cells; (ii) potentiation of signaling downstream ErbB receptors, thus contributing to tumor progression and resistance to anti-HER2 therapies; and (iii) poor clinical outcomes in women with breast cancer.

Synergistic Inhibition with a Dual Epidermal Growth Factor Receptor/HER-2/neu Tyrosine Kinase Inhibitor and a Disintegrin and Metalloprotease Inhibitor

The ErbB family of receptors is overexpressed in numerous human tumors. Overexpression correlates with poor prognosis and resistance to therapy. Use of ErbB-specific antibodies to the receptors (Herceptin or Erbitux) or ErbB-specific small-molecule inhibitors of the receptor tyrosine kinase activity (Iressa or Tarceva) has shown clinical efficacy in several solid tumors. An alternative method of affecting ErbB-initiated tumor growth and survival is to block sheddase activity. Sheddase activity is responsible for cleavage of multiple ErbB ligands and receptors, a necessary step in availability of the soluble, active form of the ligand and a constitutively activated ligand-independent receptor. This sheddase activity is attributed to the ADAM (a disintegrin and metalloprotease) family of proteins. ADAM 10 is the main sheddase of epidermal growth factor (EGF) and HER-2/neu cleavage, whereas ADAM17 is required for cleavage of additional EGF receptor (EGFR) ligands (transforming growth factor-alpha, amphiregulin, heregulin, heparin binding EGF-like ligand). This study has shown that addition of INCB3619, a potent inhibitor of ADAM10 and ADAM17, reduces in vitro HER-2/neu and amphiregulin shedding, confirming that it interferes with both HER-2/neu and EGFR ligand cleavage. Combining INCB3619 with a lapatinib-like dual inhibitor of EGFR and HER-2/neu kinases resulted in synergistic growth inhibition in MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Combining the INCB7839 second-generation sheddase inhibitor with lapatinib prevented the growth of HER-2/neu-positive BT474-SC1 human breast cancer xenografts in vivo. These results suggest that there may be an additional clinical benefit of combining agents that target the ErbB pathways at multiple points.

Intratumoral Epiregulin Is a Marker of Advanced Disease in Non–Small Cell Lung Cancer Patients and Confers Invasive Properties onEGFR-Mutant Cells

Non-small cell lung cancer (NSCLC) cells with activating epidermal growth factor receptor (EGFR) somatic mutations have unique biological properties, including high expression of the ErbB ligand epiregulin; however, the biological role of epiregulin in these cells has not been elucidated. To examine its role, we used an immunohistochemical approach to detect epiregulin expression in NSCLC biopsy samples and pharmacologic and genetic approaches to inhibit epiregulin in cultured NSCLC cells. In NSCLC biopsy samples, epiregulin was detected in 237 of 366 (64.7%) tumors, which correlated with nodal metastasis and a shorter duration of survival. In EGFR-mutant NSCLC cell lines, treatment with a small-molecule EGFR tyrosine kinase inhibitor diminished mRNA levels of the gene encoding epiregulin (EREG). The ability of EGFR-mutant NSCLC cells to invade through Matrigel in vitro was inhibited by treatment with an anti-epiregulin neutralizing antibody or by transfection with an EREG short hairpin RNA. Collectively, these findings show that epiregulin expression correlated with advanced disease, was EGFR dependent, and conferred invasive properties on NSCLC cells. Additional studies are warranted in NSCLC patients to evaluate whether epiregulin expression predicts the metastatic potential of primary tumors and whether anti-epiregulin treatment strategies are efficacious in the prevention of metastasis.

Neuregulin-mediated ErbB3 signaling is required for formation of zebrafish dorsal root ganglion neurons

Dorsal root ganglia (DRGs) arise from trunk neural crest cells that emerge from the dorsal neuroepithelium and coalesce into segmental streams that migrate ventrally along the developing somites. Proper formation of DRGs involves not only normal trunk neural crest migration, but also the ability of DRG progenitors to pause at a particular target location where they can receive DRG-promoting signals. In mammalian embryos, a receptor tyrosine kinase proto-oncogene, ErbB3, is required for proper trunk neural crest migration. Here, we show that in zebrafish mutants lacking ErbB3 function, neural crest cells do not pause at the location where DRGs normally form and DRG neurons are not generated. We also show that these mutants lack trunk neural crest-derived sympathetic neurons, but that cranial neural crest-derived enteric neurons appear normal. We isolated three genes encoding neuregulins, ErbB3 ligands, and show that two neuregulins function together in zebrafish trunk neural crest cell migration and in DRG formation. Together, our results suggest that ErbB3 signaling is required for normal migration of trunk, but not cranial, neural crest cells.

Fowler Award Presentation: Effects of ErbB2 Signaling on the Response of Vestibular Schwannoma Cells to γ‐Irradiation

AbstractObjective: For vestibular schwannomas (VSs) that require treatment, options are limited to microsurgery or irradiation (IR). Development of alternative therapies that augment or replace microsurgery or IR would benefit patients not suitable for current therapies. This study explored the ability of ErbB2 inhibitors to modulate the effects of IR on VS cells.Study Design: Prospective study using primary cultures derived from human VSs.Methods: Primary cultures of VS cells were derived from acutely resected tumors. Cultures received single escalating doses (15–40 Gy) of γ‐irradiation from a 137Cs γ‐irradiation source. Cell proliferation was determined by BrdU uptake and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Trastuzumab (Herceptin) and PD158780 were independently used to inhibit ErbB2 signaling while neuregulin‐1β (NRG‐1) was used to activate ErbB2.Results: IR induces VS cell cycle arrest and apoptosis in doses greater than 20 Gy, demonstrating that VS cells are relatively radioresistant. This radioresistance likely arises from their low proliferative capacity as a sublethal dose of IR (10 Gy) strongly induces deoxyribonucleic acid (DNA) damage evidenced by histone H2AX phosphorylation. Inhibition of ErbB2, which decreases VS cell proliferation, protects VS cells from radiation‐induced apoptosis, while NRG‐1, an ErbB2 ligand and VS cell mitogen, increases radiation‐induced VS cell apoptosis.Conclusions: Compared with many neoplastic conditions, VS cells are relatively radioresistant. The radio‐protective effect of ErbB2 inhibitors implies that the sensitivity of VS cells to IR depends on their proliferative capacity. These results hold important implications for current and future treatment strategies.

Epigenetic regulation of the human mucin gene MUC4 in epithelial cancer cell lines involves both DNA methylation and histone modifications mediated by DNA methyltransferases and histone deacetylases

The human gene MUC4 encodes a transmembrane mucin, ligand of ErbB2, that is associated with pancreatic tumor progression. In the normal pancreas, MUC4 is not expressed, whereas activation of its expression is observed in the early steps of pancreatic carcinogenesis. The molecular mechanisms responsible for MUC4 gene activation are however still unknown. The MUC4 5'-flanking region being GC-rich and including two CpG islands, we hypothesized that epigenetic regulation may be involved and undertook to decipher the molecular phenomenons implied. By treating cancer cell lines with 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA), we were able to restore MUC4 expression in a cell-specific manner. We showed by bisulfite-treated genomic DNA sequencing and chromatin immunoprecipitation that methylation of five CpG sites and establishment of a repressive histone code at the 5'-untranslated region were associated with MUC4 silencing and impaired its activation by Sp1. Direct involvement of DNMT3A, DNMT3B, HDAC1, and HDAC3 was demonstrated by RNA interference and chromatin immunoprecipitation. Moreover, inhibition of histone deacetylation by TSA was associated with strong MUC4 repression in high-expressing cells. In conclusion, this work shows for the first time the importance of epigenetics in regulating MUC4 expression and may represent a new strategy to inhibit its expression in epithelial tumors.

The Role of ErbB3 and its Binding Partners in Breast Cancer Progression and Resistance to Hormone and Tyrosine Kinase Directed Therapies

An increasingly important role for the ErbB3 receptor in the genesis and progression of breast cancer is emerging. ErbB3 is frequently overexpressed in breast cancer and coexpression of ErbB2/3 is a poor prognostic indicator. ErbB3 has also been implicated in the development of resistance to antiestrogens such as tamoxifen and ErbB tyrosine kinase inhibitors such as gefitinib. Persistent activation of the AKT pathway has been postulated to contribute to ErbB3-mediated resistance to these therapies. This activation may be due in part to the inappropriate production of the ErbB3 ligand heregulin. ErbB3 binding proteins, which negatively regulate ErbB3 protein levels and the ability of ErbB3 to transmit proliferative signals, also contribute to breast cancer progression and treatment resistance. These proteins include the intracellular RING finger E3 ubiquitin ligase Nrdp1 and the leucine-rich protein LRIG-1 that mediate receptor degradation. Ebp1, another ErbB3 binding protein, suppresses HRG driven breast cancer cell growth and contributes to tamoxifen sensitivity. These studies point to the importance of the evaluation of protein levels and functional activity of ErbB3 and its binding proteins in breast cancer prognosis and prediction of clinical response to treatment.

The Role of NRG3 in Mammary Development

The Neuregulin gene family encodes EGF-containing ligands which mediate their effects by binding to the ERBB receptor tyrosine kinases, a signalling network with important roles in both mammary gland development and breast cancer. Neuregulin3 (NRG3), a ligand for ERBB4, promotes early mammary morphogenesis and acts during specification of the mammary placode, an aggregate of epithelial cells that forms during mid-embryogenesis. Recent studies have shown that NRG3 can alter the cell fate of other epidermal progenitor populations when NRG3 is mis-expressed throughout the basal layer of the developing epidermis with the K14 promoter. Here evidence for a key function for NRG3 in promoting early mammary morphogenesis and the implication for the role of NRG3 in breast cancer and establishment of the mammary lineage are discussed.

Inhibition of heregulin mediated MCF-7 breast cancer cell growth by the ErbB3 binding protein EBP1

The ErbB2/3 heterodimer plays a critical role in breast cancer genesis and progression. EBP1, an ErbB3 binding protein, inhibits breast cancer growth but its effects on ErbB3 ligand mediated signal transduction or ErbB receptors is not known. We report here that ectopic expression of EBP1 in MCF-7 and AU565 breast cancer cell lines inhibited HRG-induced proliferation. ErbB2 protein levels were substantially decreased in EBP1 transfectants, while ErbB3 levels were unchanged. HRG-induced AKT activation was attenuated in EBP1 stable transfectants and transfection of a constitutively activated AKT partially restored the growth response to HRG. Down-regulation of EBP1 expression in MCF-7 cells by shRNA resulted in increased cell growth in response to HRG and increased cyclin D1 and ErbB2 expression. These results suggest that EBP1, by down-regulating ErbB signal transduction, attentuates HRG-mediated growth of breast cancer cells.

Common behavioral influences of the ErbB1 ligands transforming growth factor alpha and epiregulin administered to mouse neonates

Ligands for epidermal growth factor (EGF) receptor (ErbB1), such as EGF, transforming growth factor alpha (TGFα), and epiregulin, are enriched in body fluids and blood and regulate development of various peripheral organs. It remains however how such circulating polypeptide growth factors influence brain development and function. Here, we performed peripheral injections of TGFα and epiregulin to mouse neonates and evaluated immediate physical and neurochemical development and later behavioral consequences. Subcutaneous administration of TGFα and epiregulin increased phosphorylation of brain ErbB1, suggesting their effects on brain development. Repeated their injections similarly enhanced physical development of eyelid opening and tooth eruption during early postnatal stage and resulted in abnormal behavioral traits in the adult stage. Acoustic startle responses of mice treated with these growth factors as neonates were enhanced and prepulse inhibition was decreased without an apparent correlation between prepulse inhibition level and startle intensity. Locomotor activity and fear-learning performance with tone and context cues were not altered, however. These results suggest that circulating ErbB1 ligands in the periphery of neonates have some common influences on later behavioral traits. Abnormal ErbB1 ligand production at neonatal and potentially prenatal stages might therefore associate with neurodevelopmental disorders such as schizophrenia.

Phosphorylation of the ErbB3 binding protein Ebp1 by p21-activated kinase 1 in breast cancer cells

The ErbB3 binding protein (Ebp1) is a transcriptional corepressor that inhibits the activity of proliferation-associated genes and the growth of human breast cancer cell lines. Treatment of breast cancer cells with the ErbB3 ligand heregulin (HRG) results in increased phosphorylation of Ebp1 and transcriptional repression. The p21-activated serine/threonine kinase 1 (PAK1), which plays an important role in breast cancer progression and resistance to the anti-oestrogen tamoxifen, is also activated by HRG. We therefore examined the ability of PAK1 to phosphorylate and regulate the function of Ebp1. We found that PAK1 phosphorylated Ebp1 in vitro and mapped the phosphorylation site to threonine 261. Both HRG treatment and expression of a constitutively activated PAK1 in MCF-7 breast cancer cells enhanced threonine phosphorylation of Ebp1. In MCF-7 cells, ectopically expressed Ebp1 bound endogenous PAK1 and this association was enhanced by treatment with HRG. Mutation of the PAK1 phosphorylation site to glutamic acid, mimicking a phosphorylated state, completely abrogated the ability of Ebp1 to repress transcription, inhibit growth of breast cancer cell lines and contribute to tamoxifen sensitivity. These studies demonstrate for the first time that Ebp1 is a substrate of PAK1 and the importance of the PAK1 phosphorylation site for the functional activity of Ebp1 in breast cancer cells.

The ADAM10 Prodomain Is a Specific Inhibitor of ADAM10 Proteolytic Activity and Inhibits Cellular Shedding Events

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-alpha-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a K(i) of 48 nM. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer.