ErbB Ligands Research Articles

Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells.

Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z⁺ T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28⁺ T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain.

The therapeutic effect of histone deacetylase inhibitor PCI-24781 on gallbladder carcinoma in BK5.erbB2 mice

Gallbladder carcinoma (GBCa), a type of biliary tract cancer (BTC), has proven challenging to treat, demonstrating the need for more effective therapeutic strategies. In our current study, we examined the therapeutic effects of the histone deacetylase (HDAC) inhibitor PCI-24781 against GBCa that developed in BK5.erbB2 mice. PCI-24781 [50 mg/kg/day] and control solutions were administered to BK5.erbB2 mice for 4 weeks. The therapeutic effect of PCI-24781 was evaluated by ultrasound biomicroscopy (USBM) throughout the experiment and histological analyses at the end of the experiment. To investigate potential mechanisms underlining the therapeutic effects of PCI-24781 on GBCa in BK5.erbB2 mice, PCI-24781-treated gallbladders were subjected to Western blot and RT-PCR analysis. The inhibitory effect of PCI-24781 on the growth of BTC cells was compared to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and gemcitabine. To study the role of miRNAs in GBCa tumorigenesis, the expression profile of 368 miRNAs in GBCas from BK5.erbB2 (both treated and untreated) and wild type mice was analyzed. Treatment of BK5.erbB2 mice with PCI-24781 for 1 month prevented 79% of GBCa cases from progression and showed a clinical effect in 47% of cases. We also confirmed a potent inhibitory effect on tumor cell growth in human BTC cell lines treated with PCI-24781. This effect was associated with downregulation of ErbB2 mRNA and ErbB2 protein/activity and upregulation of acetylated histone and acetylated tubulin. Treatment with PCI-24781 resulted in decreased expression of Muc4, an intramembrane ligand for ErbB2, in BTC cells. PCI-24781 had more effects on growth inhibition of BTC cells than SAHA. In addition, PCI-24781 effectively inhibited the growth of gemcitabine-resistant cells. miRNA profiling revealed that the expression of several miRNAs was significantly altered in GBCa in the BK5.erbB2 mouse compared to normal gallbladder, including upregulated miR21, which was downregulated by PCI-24781. These results indicate that PCI-24781 potently inhibits the growth of BTC cells by decreasing ErbB2 expression and activity as well as regulating altered miRNA expression. PCI-24781 may have a potential value as a novel chemotherapeutic agent against human BTC in which ErbB2 is overexpressed.

Exogenous GLP-2 and IGF-I induce a differential intestinal response in IGF binding protein-3 and -5 double knockout mice

Glucagon-like peptide-2 (GLP-2) action is dependent on intestinal expression of IGF-I, and IGF-I action is modulated by IGF binding proteins (IGFBP). Our objective was to evaluate whether the intestinal response to GLP-2 or IGF-I is dependent on expression of IGFBP-3 and -5. Male, adult mice in six treatment groups, three wild-type (WT) and three double IGFBP-3/-5 knockout (KO), received twice daily intraperitoneal injections of GLP-2 (0.5 μg/g body wt), IGF-I (4 μg/g body wt), or PBS (vehicle) for 7 days. IGFBP-3/-5 KO mice showed a phenotype of lower plasma IGF-I concentration, but greater body weight and relative mass of visceral organs, compared with WT mice (P < 0.001). WT mice showed jejunal growth with either IGF-I or GLP-2 treatment. In KO mice, IGF-I did not stimulate jejunal growth, crypt mitosis, sucrase activity, and IGF-I receptor (IGF-IR) expression, suggesting that the intestinotrophic actions of IGF-I are dependent on expression of IGFBP-3 and -5. In KO mice, GLP-2 induced significant increases in jejunal mucosal cellularity, crypt mitosis, villus height, and crypt depth that was associated with increased expression of the ErbB ligand epiregulin and decreased expression of IGF-I and IGF-IR. This suggests that in KO mice, GLP-2 action in jejunal mucosa is independent of the IGF-I system and linked with ErbB ligands. In summary, the intestinotrophic actions of IGF-I, but not GLP-2, in mucosa are dependent on IGFBP-3 and -5. These findings support the role of multiple downstream mediators for the mucosal growth induced by GLP-2.

A Notch1–neuregulin1 autocrine signaling loop contributes to melanoma growth

The Notch pathway is an evolutionary conserved signaling cascade that has an essential role in melanoblast and melanocyte stem cell homeostasis. Notch signaling is emerging as a key player in melanoma, the most deadly form of skin cancer. In melanoma, Notch1 is inappropriately reactivated and contributes to melanoma tumorigenicity. Here, we propose a novel mechanism by which Notch1 promotes the disease. We found that Notch1 directly regulates the transcription of neuregulin1 (NRG1) by binding to its promoter region. NRG1 is the ligand for ERBB3 and 4, members of the epidermal growth factor family of receptors that are involved in the genesis and progression of a number of cancers. Notch1 and NRG1 expression are associated in melanoma and inhibition of NRG1 signaling leads to melanoma cell growth inhibition and tumor growth delay. Mechanistically, these effects are associated with the inhibition of the PI3Kinase/Akt signaling pathway and with the accumulation of p27(Kip1). On the other end, addition of recombinant NRG1 can partially restore melanoma cell growth that is inhibited by Notch1 ablation. Taken together, our findings underline a new, previously undescribed autocrine signaling loop between Notch1 and NRG1 that controls melanoma growth and provide experimental evidence that the targeting of Notch and ERBB signaling may represent a novel potential therapeutic approach in melanoma.

Novel Nonphosphorylated Peptides with Conserved Sequences Selectively Bind to Grb7 SH2 Domain with Affinity Comparable to Its Phosphorylated Ligand

The Grb7 (growth factor receptor-bound 7) protein, a member of the Grb7 protein family, is found to be highly expressed in such metastatic tumors as breast cancer, esophageal cancer, liver cancer, etc. The src-homology 2 (SH2) domain in the C-terminus is reported to be mainly involved in Grb7 signaling pathways. Using the random peptide library, we identified a series of Grb7 SH2 domain-binding nonphosphorylated peptides in the yeast two-hybrid system. These peptides have a conserved GIPT/K/N sequence at the N-terminus and G/WD/IP at the C-terminus, and the region between the N-and C-terminus contains fifteen amino acids enriched with serines, threonines and prolines. The association between the nonphosphorylated peptides and the Grb7 SH2 domain occurred in vitro and ex vivo. When competing for binding to the Grb7 SH2 domain in a complex, one synthesized nonphosphorylated ligand, containing the twenty-two amino acid-motif sequence, showed at least comparable affinity to the phosphorylated ligand of ErbB3 in vitro, and its overexpression inhibited the proliferation of SK-BR-3 cells. Such nonphosphorylated peptides may be useful for rational design of drugs targeted against cancers that express high levels of Grb7 protein.

Expression changes of the neuregulin 1 isoforms in neuropathic pain model rats

The neuregulin1 ( Nrg1) gene that is expressed in the dorsal root ganglion (DRG) contains an EGF-like domain, which is known to be a direct ligand for ErbB3 and ErbB4. Multiple splice variants of the Nrg1 gene are broadly classified into 3 groups by structural features (type I, type II and type III) and their functions differ in various tissues. The Nrg1 gene has emerged as a key mediator of axon–Schwann cell interactions and as a regulator of Schwann cell development. The Nrg1 gene is indicated as a promising growth factor for neuronal development. However, the function of the Nrg1 in pain has not been clarified. We therefore, examined the expression profiles of each type of the Nrg1 transcript in the bilateral L4/L5 DRGs using L5 spinal nerve ligation (SNL) model rats and complete Freund's adjuvant (CFA) model rats. Behavior tests have shown typical mechanical hyperalgesia in both the L5SNL model and the CFA model. In the L5SNL model, expression of the Nrg1 type I and type II were significantly increased in the L5 DRG. On the other hand, the expression of the Nrg1 type III was decreased in the L5 DRG. We demonstrated that the expression changes of the Nrg1 isoforms in the ipsilateral DRGs were preferentially related to the response to nerve injury. Our findings suggest that the aberrant expression may play an important role in nerve injury, regeneration and subsequent neuropathic pain on the L5SNL.

P1-02-01: c-Jun N-Terminal Kinase 1 (JNK1) Inhibits Tumor Growth and Metastasis by Downregulating Epithelial to Mesenchymal Transition (EMT) and Stem Cell-Related Genes.

Abstract Murine and human mammary cancers often show dysregulation of important signaling pathways including the canonical Wingless-iNTegrated (Wnt) and avian ERythroBlastosis oncogene B (ErbB) pathways. Transgenic expression of Wnt ligands causes transformation of normal mammary cells in mice, and Wnt10b is frequently upregulated in human breast cancers. Overexpression of ErbB ligands and amplification of receptors have also been implicated in human breast cancer. JNK1 is a tumor suppressor in the skin and intestinal epithelium, and JNKs are known to integrate ErbB and Wnt pathways as well as others to control cell growth and differentiation. In a murine mammary cancer model where 4T1.2 cells were injected into the mammary gland, reducing JNK1 levels by expressing shRNA (shJNK1) resulted in increased tumor growth and lung metastasis compared to mice injected with control vector- (pSM2) expressing cells. A microarray analysis comparing gene expression between shJNK1 and control tumors revealed 2 and 2.5-fold increases in the ErbB pathway genes Nrg3 and ETV5, 2-fold increases in the Wnt genes Bcl-9 and Wnt10a, and a 1.6-fold increase in the EMT gene Twist1. RT-PCR analysis of in vitro grown 4T1.2 cell lines transfected with shJNK1 confirmed increased expression of Bcl-9 and ETV5, as well as the ETV5 target Cox-2. ErbB2 protein was also overexpressed. The shJNK1 cells showed upregulation of pERK in response to Heregulin1 (an Erbb2/3 ligand) and Fibroblast Growth Factor (FGF) 1, which further amplifies canonical Wnt signaling. In a p53-/− tumor model, ETV5, Bcl-9, and Cox-2 were still upregulated in jnk1-/−compared to wildtype tumor cells, indicating this effect is p53-independent. In the normal mammary gland, a 4-fold increase in ETV5 and a 5-fold increase in Twist1 were found in jnk1-/− mice compared to wildtype, further indicating that this effect is dependent on JNK1 alone. The ErbB and Wnt pathways are known to upregulate EMT and stem-cell related genes, however, the involvement of JNK1 in these effects is a novel hypothesis. Thus far, our data suggest that JNK1 deficiency targets these oncogenic pathways to contribute to a more aggressive tumor phenotype due to heightened EMT and “stem-cellness”. Further studies using cell sorting and differentiation assays will determine whether normal jnk1-/− glands contain a higher fraction of stem cells than wildtype glands. Inhibition of EMT genes and/or ETV5 in the p53-/−;jnk1-/− cancer cells will determine whether the tumor growth or metastasis phenotypes are dependent on these genes. EMT and stem-cell genes are frequently expressed in human breast cancer subtypes that exhibit low survival rates, and EMT is known to be linked to increased metastasis. Some of these sub-types, such as claudin-low tumors, currently have no molecularly-targeted treatments, therefore it is important to determine what proteins critically contribute to these phenotypes so that efficient and effective treatments can be developed. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-01.

A recombinant decoy comprising EGFR and ErbB-4 inhibits tumor growth and metastasis

EGF-like growth factors control tumor progression, as well as evasion from the toxic effects of chemotherapy. Accordingly, antibodies targeting the cognate receptors, such as EGFR/ErbB-1 and the co-receptor HER2/ErbB-2, are widely used to treat cancer patients, but agents that target the EGF-like growth factors are not available. To circumvent the existence of 11 distinct ErbB ligands, we constructed a soluble fusion protein (hereinafter: TRAP-Fc) comprising truncated extracellular domains of EGFR/ErbB-1 and ErbB-4. The recombinant TRAP-Fc retained high affinity ligand binding to EGF-like growth factors and partially inhibited growth of a variety of cultured tumor cells. Consistently, TRAP-Fc displayed an inhibitory effect in xenograft models of human cancer, as well as synergy with chemotherapy. Additionally, TRAP-Fc inhibited invasive growth of mammary tumor cells and reduced their metastatic seeding in the lungs of animals. Taken together, the activities displayed by TRAP-Fc reinforce critical roles of EGF-like growth factors in tumor progression, and they warrant further tests of TRAP-Fc in pre-clinical models.

Abstract C58: Humanization and characterization of an anti-ErbB-3 murine monoclonal antibody.

Abstract Background: The signaling network downstream of the human epidermal growth factor receptors ErbB-1 and ErbB-2 have been extensively targeted by cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance which is often associated with reactivation of the ErbB-3/PI3K-Akt axis. This may be overcome by ErbB-3 ligands that abrogates receptor-mediated signaling. Methods: the murine MP-RM-1 monoclonal antibody that specifically binds ErbB-3 ectodomain was chimerized by replacing the murine constant regions with the human constant regions and humanized by gratfing Complementarity Determining Regions from the murine into a human antibody framework using a computer assisted molecular modelling. Recombinant genes were placed into the pCDNA3.1 expression vector and transfected into Chinese Hamster Ovary-S cells. Antibodies were screened on the basis of their ability to inhibit Neuregulin-1 (NRG-1)-induced activation of the ErbB-3/PI3K-Akt axis in IR-8 human melanoma cells (Table 1). Four antibody variants, i.e., cMP-RM-1 #1, hMP-RM-1 #6, hMP-RM-1 #10, and hMP-RM-1 #20, were selected and further tested for their ability to promote ErbB-3 downregulation and to inhibit in vitro and in vivo growth of tumor cells. Results: Four chimeric (c) and sixteen humanized (h) antibody variants were obtained. All variants showed to possess antigen binding affinity comparable to that of their parental antibody. Initial screening identified one chimeric (cMP-RM-1 #1) and three humanized (hMP-RM-1 #6, hMP-RM-1 #10, hMP-RM-1 #20) antibodies as the most effective in inhibiting ErbB-3/Akt phosphorylation and promotion of ErbB-3 downregulation. Further testing revealed the antibodies potently inhibited colony formation ability of tumor cells and halted growth of melanoma and ovarian cancer xenografts in nude mice. Treatment with the antibodies was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Conclusions: This successful production of chimeric/humanized antibodies possessing antitumor activity in vitro and in vivo may provide novel candidates for ErbB-3-targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C58.

Abstract C118: Optimization of MM-111 and lapatinib dosing regimens using mathematical modeling and quantitative biology.

Abstract Designing therapeutically optimal dosing regimens for targeted biologic therapies is an open problem in translational oncology. There are at least two underlying reasons for this problem: First, biologics are generally well tolerated so it is not guaranteed that the maximum tolerated dose (MTD) will be observed in a Phase 1 dose escalation; and second, since targeted therapies work in only a subset of patients, there will be limited efficacy data available to determine a Phase 2 dose. This problem is further compounded by the use of combination drug treatments. MM-111 is a novel bispecific antibody against ErbB3 (HER3), using an ErbB2 (HER2) targeting arm to enhance avidity and inhibitor potency. Pre-clinical data shows that combining MM-111 with the ErbB2 inhibitor lapatinib (Tykerb®) results in synergistic tumor growth inhibition. We desired to leverage our pre-clinical modeling, signaling, pharmacokinetic (PK), pharmacodynamic (PD), and efficacy data to optimize dosing regimens of the combination therapy. While this study design is focused on MM-111 combined with lapatinib, this strategy is generally applicable to molecular targeted therapeutics given alone or in combination. Our approach was to integrate a mathematical model of the MM-111 and lapatinib's mechanism of action, incorporating ErbB-family signal transduction, transcriptional regulation, and resulting tumor proliferation, with pre-clinical PK models of the two drugs. This integrated model was trained using in vitro dose response data from BT-474-M3 cells treated with the ErbB3 ligand heregulin plus combinations of the two drugs. Levels of phospho- and total ErbB3, ERbB2, AKT, ERK, and p27 were quantified using ELISA, and cell growth effects quantified using the Cell Titer Glow assay. This was then integrated with a 2-compartment PK-model, to predict PD profiles and tumor growth effects in murine xenografts treated with the drug combinations. After validation of our model with these initial in vivo studies, we designed therapeutically optimal dosing schedules in silico. The novel dosing strategies are now being validated in xenografts, and will help inform future clinical studies. By integrating PK/PD with known molecular mechanisms of action, we are able to rationally design combination treatment strategies to maximize therapeutic synergy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C118.

Monosynaptic inputs to ErbB4‐expressing inhibitory neurons in mouse primary somatosensory cortex

Previous reports have described inputs to the somatosensory cortex (S1) in mouse or rat using retrograde or anterograde tracers. Such studies do not, however, reveal which particular cell types within the S1 cortex receive direct monosynaptic connections from these input sources. Here we describe the monosynaptic inputs to a subpopulation of mouse S1 inhibitory neurons that express ErbB4. We used a previously described "bridge protein," composed of the ErbB4 ligand, neuregulin (NRG1), fused to the avian viral receptor TVB (TVB-NRG1), along with EnvB pseudotyped lentivirus (LV) and rabies virus (RV), to selectively coinfect ErbB4-expressing neurons (Choi et al. [2010] Proc Natl Acad Sci U S A 107:16703-16708). The RV had its glycoprotein gene deleted and replaced with mCherry, so that infected cells express mCherry and the virus cannot spread without provision of rabies glycoprotein (RG) by transcomplementation. The LV encoded and expressed RG to allow transcomplementation in coinfected neurons, so that the RV could spread transsynaptically and label their direct monosynaptic inputs. The RV could not spread beyond the direct inputs, due to the lack of RG in presynaptic cells. This method revealed long-range connections from thalamus, nucleus basalis, raphe, and distant cortical areas, including ipsilateral motor, secondary somatosensory, retrosplenial, and perirhinal cortex and contralateral S1. In addition, local connections from ipsilateral pyramidal neurons within S1 were labeled. These input sources account for all of the known inputs to S1 described with standard tracers, suggesting that the subpopulation of ErbB4-positive inhibitory neurons infected using the TVB-NRG1 bridge protein receives inputs indiscriminately from S1 input sources.

Translational research of a novel humanized epidermal growth factor receptor-related protein: a putative inhibitor of pan-ErbB

The ErbB family members are protein tyrosine kinases, which play a crucial role in the signal transduction pathways that regulate key cellular functions. Overexpression of the ErbB family members is associated with oncogenicity, metastatic potential, cell proliferation, apoptosis, angiogenesis, and prognosis in cancer. Molecular-targeted therapies centered on the ErbB signaling pathway are the currently promising anti-cancer therapies. We reviewed the literature to summarize the current knowledge of epidermal growth factor receptor (EGFR)-related protein (ERRP) and determine the potential of this protein to be translated into a molecular-targeting treatment for cancer. ERRP isolated from rat gastroduodenal mucosa is a pan-ErbB inhibitor that targets multiple members of the ErbB family both in vitro and in vivo. Sequestration of ErbB ligands by ERRP results in the formation of inactive ErbB heterodimers and subsequent attenuation of signaling pathways activated by ErbB. We suggest a strategy to develop a humanized ERRP protein based on activity of rat EERP in vitro. As rat ERRP protein is expected to generate an immune response in humans, we propose a hypothesis that a humanized version of ERRP has potential therapeutic value for cancer patients.

Expression and function of ErbB receptors and ligands in the pituitary

The role of ErbB family in discreet pituitary functions is reviewed. Several ErbB receptor ligands, EGF, TGFα, and heregulin are differentially expressed in normal gonadotroph and lacto-somatotroph lineages, and other elements of the anterior pituitary. ErbB receptors, i.e. EGFR and ErbB2, are also localized to the anterior pituitary with preferential EGFR lactosomatotroph expression. EGF regulates CRH and ACTH secretion and corticotroph proliferation as well as exhibiting autocrine and paracrine effects on gonadotrophs and on lactosomatotroph proliferation, gene and protein expression, and hormonal secretion. EGF and EGFR are expressed in both functioning and non-functioning pituitary adenomas, with higher expression in more aggressive tumor subtypes. ErbB2 receptor is detected in all tumor subtypes, particularly in invasive tumors. ErbB tyrosine kinase inhibitors regulate hormonal secretion, cell morphology, and proliferation in lacto-somatotroph tumors, reflecting the emerging application of targeted pituitary therapeutics.

A multivariate model of ErbB network composition predicts ovarian cancer cell response to canertinib

Identifying the optimal treatment strategy for cancer is an important challenge, particularly for complex diseases like epithelial ovarian cancer (EOC) that are prone to recurrence. In this study we developed a quantitative, multivariate model to predict the extent of ovarian cancer cell death following treatment with an ErbB inhibitor (canertinib, CI-1033). A partial least squares regression model related the levels of ErbB receptors and ligands at the time of treatment to sensitivity to CI-1033. In this way, the model mimics the clinical problem by incorporating only information that would be available at the time of drug treatment. The full model was able to fit the training set data and was predictive. Model analysis demonstrated the importance of including both ligand and receptor levels in this approach, consistent with reports of the role of ErbB autocrine loops in EOC. A reduced multi-protein model was able to predict CI-1033 sensitivity of six distinct EOC cell lines derived from the three subtypes of EOC, suggesting that quantitatively characterizing the ErbB network could be used to broadly predict EOC response to CI-1033. Ultimately, this systems biology approach examining multiple proteins has the potential to uncover multivariate functions to identify subsets of tumors that are most likely to respond to a targeted therapy.

An ErbB-3 antibody, MP-RM-1, inhibits tumor growth by blocking ligand-dependent and independent activation of ErbB-3/Akt signaling

The ErbB receptors, such as ErbB-1 and ErbB-2, have been intensely pursued as targets for cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance, which is often associated with reactivation of the ErbB-3-PI3K-Akt signaling. This may be overcome by an ErbB-3 ligand that abrogates receptor-mediated signaling. Toward this end, we have generated a mouse monoclonal antibody, MP-RM-1, against the extracellular domain (ECD) of ErbB-3 receptor. Assessment of human tumor cell lines, as well as early passage tumor cells revealed that MP-RM-1 effectively inhibited both NRG-1β-dependent and -independent ErbB-3 activation. The antagonizing effect of MP-RM-1 was of non-competitive type, as binding of [(125)I]-labeled NRG-1β to ErbB-3 was not influenced by the antibody. MP-RM-1 treatment led, in most instances, to decreased ErbB-3 expression. In addition, MP-RM-1 was able to inhibit the colony formation ability of tumor cells and tumor growth in two human tumor xenograft nude mouse models. Treatment with the antibody was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Collectively, these results indicate that MP-RM-1 has the potential to interfere with signaling by ErbB-3 and reinforce the notion that ErbB-3 could be a key target in cancer-drug design.

ErbB2 Is Necessary for ErbB4 Ligands to Stimulate Oncogenic Activities in Models of Human Breast Cancer

ErbB4 is a member of the ErbB family of receptor tyrosine kinases. This family includes ErbB2 (HER2/Neu), a validated therapeutic target in breast cancer. Several studies indicate that ErbB4 functions as a tumor suppressor in breast cancer, whereas others indicate that ErbB4 functions as an oncogene. Here the authors explore the context in which ErbB4 functions as an oncogene. Silencing expression of either ErbB2 or ErbB4 in breast tumor cell lines results in reduced stimulation of anchorage independence and cell motility by the ErbB4 agonist neuregulin 2β. ErbB2 tyrosine kinase activity, but not ErbB4 tyrosine kinase activity, is required for neuregulin 2β to stimulate cell proliferation. Moreover, sites of ErbB4 tyrosine phosphorylation, but not sites of ErbB2 tyrosine phosphorylation, are required for neuregulin 2β to couple to cell proliferation. These data suggest that targeting ErbB2 expression or tyrosine kinase activity may be effective in treating ErbB4-dependent breast tumors, even those tumors that lack ErbB2 overexpression.

Function of erbB receptors and DNA-PKcs on phosphorylation of cytoplasmic and nuclear Akt at S473 induced by erbB1 ligand and ionizing radiation

Background and purposeIn the present study effect of erbB2 as well as DNA-PKcs on ionizing radiation (IR)- and erbB1 ligand-induced phosphorylation of Akt at S473 in cytoplasmic and nuclear fractions was investigated. Materials and methodsDNA-PKcs proficient and deficient syngeneic colon carcinoma sublines of HCT116 and the glioblastoma cell lines MO59K and MO59J as well as the lung carcinoma cell line A549 were used. Akt-S473 phosphorylation was investigated in cells pre-treated with pharmacological inhibitors or transfected with siRNA by immunoprecipitation, Western blotting and confocal microscopy after different stimuli, i.e., ligands and IR. ResultsIR-induced phosphorylation of Akt in both MO59K and MO59J cell lines but not in HCT116 cells was DNA-PKcs dependent. In A549 cells, IR-induced phosphorylation of nuclear Akt-S473 was dependent on erbB1, erbB2, and DNA-PKcs. EGF induced phosphorylation of nuclear Akt-S473 in a DNA-PKcs and erbB2 independent manner. ConclusionData indicate that the function of DNA-PKcs on IR-induced Akt-S473 phosphorylation is cell line specific. IR-induced, but not EGF-induced phosphorylation of cytoplasmic and/or nuclear Akt-S473 is erbB2 dependent.

TNF-α converting enzyme-mediated ErbB4 transactivation by TNF promotes colonic epithelial cell survival.

Disruption of intestinal epithelial homeostasis, including enhanced apoptosis, is a hallmark of inflammatory bowel disease (IBD). We have recently shown that tumor necrosis factor (TNF) increases the kinase activity of ErbB4, a member of the epidermal growth factor receptor family that is elevated in mucosa of IBD patients and that promotes colon epithelial cell survival. In this study, we tested the hypothesis that TNF transactivates ErbB4 through TNF-α converting enzyme (TACE)-mediated ligand release and that this transactivation is necessary to protect colonic epithelial cells from cytokine-induced apoptosis. Using neutralizing antibodies, we show that heparin-binding EGF-like growth factor (HB-EGF) is required for ErbB4 phosphorylation in response to TNF. Pharmacological or genetic inhibition of the metalloprotease TACE, which mediates HB-EGF release from cells, blocked TNF-induced ErbB4 activation. MEK, but not Src or p38, was also required for transactivation. TACE activity and ligand binding were required for ErbB4-mediated antiapoptotic signaling; whereas mouse colon epithelial cells expressing ErbB4 were resistant to TNF-induced apoptosis, TACE inhibition or blockade of ErbB4 ligand binding reversed the survival advantage. We conclude that TNF transactivates ErbB4 through TACE-dependent HB-EGF release, thus protecting colon epithelial cells from cytokine-induced apoptosis. These findings have important implications for understanding how ErbB4 protects the colon from apoptosis-induced tissue injury in inflammatory conditions such as IBD.

Effects of an ErbB-3 antibody, MP-RM-1, on tumor growth and ligand-dependent and -independent activation of ErbB-3/akt signaling.

e13538 Background: The ErbB receptors, such as ErbB-1 and ErbB-2, have been intensely pursued as targets for cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance which is often associated with reactivation of the ErbB-3-PI3K-Akt signaling. This may be overcome by an ErbB-3 ligand binding molecule that abrogates ErbB-3 mediated signaling. Methods: we have generated a mouse monoclonal antibody, MP-RM-1, against the extracellular domain (ECD) of ErbB-3 receptor. The ability of the antibody to suppress NRG-1b-dependent and independent ErbB-3 signaling was evaluated in vitro by western blotting using a panel of human tumor cell lines (breast, melanoma, stomach and prostate) as well as early passage tumor cells obtained from patients. The effect on tumor growth in vitro was evaluated with clonogenic assay and in vivo using human tumor xenograft nude mouse models. Results: MP-RM-1 effectively inhibited NRG-1b stimulated and basal ErbB-3/Akt activation. Treatment with the antibody led, in most instances, to a decreased ErbB-3 expression. In addition, MP-RM-1 was able to inhibit the colony formation ability of tumor cells and tumor growth in human melanoma and prostate carcinoma xenografts. Tumors from animals treated with the antibody displayed a lower ErbB-3/Akt phosphorylation that was accompanied by a decreased ErbB-3 expression. Conclusions: MP-RM-1 has the potential to interfere with signaling by ErbB-3/Akt signaling and can be regarded as a potential candidate for clinical development.

Qualitative and quantitative re‐evaluation of epidermal growth factor‐ErbB1 action on developing midbrain dopaminergic neurons in vivo and in vitro: target‐derived neurotrophic signaling (Part 1)

Although epidermal growth factor (EGF) receptor (ErbB1) is implicated in Parkinson's disease and schizophrenia, the neurotrophic action of ErbB1 ligands on nigral dopaminergic neurons remains controversial. Here, we ascertained colocalization of ErbB1 and tyrosine hydroxylase (TH) immunoreactivity and then characterized the neurotrophic effects of ErbB1 ligands on this cell population. In mesencephalic culture, EGF and glial-derived neurotrophic factor (GDNF) similarly promoted survival and neurite elongation of dopaminergic neurons and dopamine uptake. The EGF-promoted dopamine uptake was not inhibited by GDNF-neutralizing antibody or TrkB-Fc, whereas EGF-neutralizing antibody fully blocked the neurotrophic activity of the conditioned medium that was prepared from EGF-stimulated mesencephalic cultures. The neurotrophic action of EGF was abolished by ErbB1 inhibitors and genetic disruption of erbB1 in culture. In vivo administration of ErbB1 inhibitors to rat neonates diminished TH and dopamine transporter (DAT) levels in the striatum and globus pallidus but not in the frontal cortex. In parallel, there was a reduction in the density of dopaminergic varicosities exhibiting intense TH immunoreactivity. In agreement, postnatal erbB1-deficient mice exhibited similar decreases in TH levels. Although neurotrophic supports to dopaminergic neurons are redundant, these results confirm that ErbB1 ligands contribute to the phenotypic and functional development of nigral dopaminergic neurons.