Diana Graus-Porta

ErbB‐2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling

We have analyzed ErbB receptor interplay induced by the epidermal growth factor (EGF)‐related peptides in cell lines naturally expressing the four ErbB receptors. Down‐regulation of cell surface ErbB‐1 or ErbB‐2 by intracellular expression of specific antibodies has allowed us to delineate the role of these receptors during signaling elicited by: EGF and heparin binding EGF (HB‐EGF), ligands of ErbB‐1; betacellulin (BTC), a ligand of ErbB‐1 and ErbB‐4; and neu differentiation factor (NDF), a ligand of ErbB‐3 and ErbB‐4. Ligand‐induced ErbB receptor heterodimerization follows a strict hierarchy and ErbB‐2 is the preferred heterodimerization partner of all ErbB proteins. NDF‐activated ErbB‐3 or ErbB‐4 heterodimerize with ErbB‐1 only when no ErbB‐2 is available. If all ErbB receptors are present, NDF receptors preferentially dimerize with ErbB‐2. Furthermore, EGF‐ and BTC‐induced activation of ErbB‐3 is impaired in the absence of ErbB‐2, suggesting that ErbB‐2 has a role in the lateral transmission of signals between other ErbB receptors. Finally, ErbB‐1 activated by all EGF‐related peptides (EGF, HB‐EGF, BTC and NDF) couples to SHC, whereas only ErbB‐1 activated by its own ligands associates with and phosphorylates Cbl. These results provide the first biochemical evidence that a given ErbB receptor has distinct signaling properties depending on its dimerization.

ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer.

Overexpression of the erbB‐2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB‐2‐encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB‐2 has been reported. We show that in various cells ErbB‐2 can form heterodimers with both EGF receptor (ErbB‐1) and NDF receptors (ErbB‐3 and ErbB‐4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB‐2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB‐2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum‐trapped antibody. We report that ErbB‐2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB‐2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c‐Jun kinase (SAPK), by either ligand. Our results imply that ErbB‐2 is a pan‐ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB‐2 in human cancers may be due to its ability to potentiate in trans growth factor signaling.

β1-Class Integrins Regulate the Development of Laminae and Folia in the Cerebral and Cerebellar Cortex

Mice that lack all beta1-class integrins in neurons and glia die prematurely after birth with severe brain malformations. Cortical hemispheres and cerebellar folia fuse, and cortical laminae are perturbed. These defects result from disorganization of the cortical marginal zone, where beta1-class integrins regulate glial endfeet anchorage, meningeal basement membrane remodeling, and formation of the Cajal-Retzius cell layer. Surprisingly, beta1-class integrins are not essential for neuron-glia interactions and neuronal migration during corticogenesis. The phenotype of the beta1-deficient mice resembles pathological changes observed in human cortical dysplasias, suggesting that defective integrin-mediated signal transduction contributes to the development of some of these diseases.

Single-Chain Antibody-Mediated Intracellular Retention of ErbB-2 Impairs Neu Differentiation Factor and Epidermal Growth Factor Signaling

ErbB-2 becomes rapidly phosphorylated and activated following treatment of many cell lines with epidermal growth factor (EGF) or Neu differentiation factor (NDF). However, these factors do not directly bind ErbB-2, and its activation is likely to be mediated via transmodulation by other members of the type I/EGF receptor (EGFR)-related family of receptor tyrosine kinases. The precise role of ErbB-2 in the transduction of the signals elicited by EGF and NDF is unclear. We have used a novel approach to study the role of ErbB-2 in signaling through this family of receptors. An ErbB-2-specific single-chain antibody, designed to prevent transit through the endoplasmic reticulum and cell surface localization of ErbB-2, has been expressed in T47D mammary carcinoma cells, which express all four known members of the EGFR family. We show that cell surface expression of ErbB-2 was selectively suppressed in these cells and that the activation of the mitogen-activated protein kinase pathway and p70/p85S6K, induction of c-fos expression, and stimulation of growth by NDF were dramatically impaired. Activation of mitogen-activated protein kinase and p70/p85S6K and induction of c-fos expression by EGF were also significantly reduced. We conclude that in T47D cells, ErbB-2 is a major NDF signal transducer and a potentiator of the EGF signal. Thus, our observations demonstrate that ErbB-2 plays a central role in the type I/EGFR-related family of receptors and that receptor transmodulation represents a crucial step in growth factor signaling.

ErbB-1 and ErbB-2 Acquire Distinct Signaling Properties Dependent upon Their Dimerization Partner

ABSTRACT The different epidermal growth factor (EGF)-related peptides elicit a diverse array of biological responses as the result of their ability to activate distinct subsets of ErbB receptor dimers, leading to the recruitment of different intracellular signaling networks. To specifically examine dimerization-dependent modulation of receptor signaling, we constructed NIH 3T3 cell lines expressing ErbB-1 and ErbB-2 singly and in pairwise combinations with each other ErbB family member. This model system allowed the comparison of EGF-activated ErbB-1 with ErbB-1 activated by Neu differentiation factor (NDF)-induced heterodimerization with ErbB-4. In both cases, ErbB-1 coupled to the adaptor protein Shc, but only when activated by EGF was it able to interact with Grb2. Compared to the rapid internalization of EGF-activated ErbB-1, NDF-activated ErbB-1 showed delayed internalization characteristics. Furthermore, the p85 subunit of phosphatidylinositol kinase (PI3-K) associated with EGF-activated ErbB-1 in a biphasic manner, whereas association with ErbB-1 transactivated by ErbB-4 was monophasic. The signaling properties of ErbB-2 following heterodimerization with the other ErbB receptors or homodimerization induced by point mutation or monoclonal antibody treatment were also analyzed. ErbB-2 binding to peptides containing the Src homology 2 domain of Grb2 or p85 and the phosphotyrosine binding domain of Shc varied according to the mode of receptor activation. Finally, tryptic phosphopeptide mapping of both ErbB-1 and ErbB-2 revealed that receptor phosphorylation is dependent on the dimerization partner. Differential receptor phosphorylation may, therefore, be the basis for the differences in the signaling properties observed.

Reelin, Disabled 1, and β1 integrins are required for the formation of the radial glial scaffold in the hippocampus

The extracellular matrix molecule Reelin is required for the correct positioning of neurons during the development of the forebrain. However, the mechanism of Reelin action on neuronal migration is poorly understood. Reelin is assumed to act on neurons directly, but it may also affect the differentiation of glial cells necessary for neuronal migration. Here we show that a regular glial scaffold fails to form in vivo in the dentate gyrus of mice deficient of Reelin or Disabled 1, a neuronal adaptor protein in the Reelin signaling pathway. A subset of these defects is observed in mice that lack β1-class integrins, known to bind Reelin. Moreover, recombinant Reelin induced branching of glial processes in vitro. Our data suggest that Reelin affects glial differentiation via Disabled 1 and β1-class integrin-dependent signaling pathways.

Neu differentiation factor activation of ErbB-3 and ErbB-4 is cell specific and displays a differential requirement for ErbB-2.

Neu differentiation factor (NDF)-induced signaling involves the activation of members of the ErbB family of receptor tyrosine kinases. Although ectopic expression of recombinant ErbB receptors has yielded valuable insight into their signaling properties, the biological function and in vivo interplay of these receptors are still poorly understood. We addressed this issue by studying NDF signaling in various human cell lines expressing moderate levels of all known ErbB receptors. NDF-induced phosphorylation of ErbB-2 and ErbB-3 was found in the breast epithelial cell line MCF10A, the breast tumor cell lines T47D and MCF7, and the ovarian tumor cell line OVCAR3. Despite similar expression levels, NDF-induced phosphorylation of ErbB-4 was cell specific and only detected in T47D and OVCAR3 cells. Blocking cell surface expression of ErbB-2 by intracellular expression of a single-chain antibody revealed that in these two cell lines, ErbB-2 significantly enhanced phosphorylation of ErbB-4. Efficient NDF-induced phosphorylation of ErbB-3 was strictly ErbB-2 dependent in the breast tumor cell lines T47D and MCF7, while it was largely ErbB-2 independent in MCF10A and OVCAR3 cells. Consequently, NDF-stimulated intracellular signaling and induction of a biological response displayed a cell-specific requirement for ErbB-2. Thus, while ErbB-2 cooperates with NDF receptors in the breast tumor cell lines, ErbB-2 independent mechanisms seem to prevail in other cellular contexts.

FGFR Genetic Alterations Predict for Sensitivity to NVP-BGJ398, a Selective Pan-FGFR Inhibitor

UNLABELLED Patient stratification biomarkers that enable the translation of cancer genetic knowledge into clinical use are essential for the successful and rapid development of emerging targeted anticancer therapeutics. Here, we describe the identification of patient stratification biomarkers for NVP-BGJ398, a novel and selective fibroblast growth factor receptor (FGFR) inhibitor. By intersecting genome-wide gene expression and genomic alteration data with cell line-sensitivity data across an annotated collection of cancer cell lines called the Cancer Cell Line Encyclopedia, we show that genetic alterations for FGFR family members predict for sensitivity to NVP-BGJ398. For the first time, we report oncogenic FGFR1 amplification in osteosarcoma as a potential patient selection biomarker. Furthermore, we show that cancer cell lines harboring FGF19 copy number gain at the 11q13 amplicon are sensitive to NVP-BGJ398 only when concomitant expression of β-klotho occurs. Thus, our findings provide the rationale for the clinical development of FGFR inhibitors in selected patients with cancer harboring tumors with the identified predictors of sensitivity. SIGNIFICANCE The success of a personalized medicine approach using targeted therapies ultimately depends on being able to identify the patients who will benefit the most from any given drug. To this end, we have integrated the molecular profiles for more than 500 cancer cell lines with sensitivity data for the novel anticancer drug NVP-BGJ398 and showed that FGFR genetic alterations are the most significant predictors for sensitivity. This work has ultimately endorsed the incorporation of specific patient selection biomakers in the clinical trials for NVP-BGJ398.

β1-Integrins Are Critical for Cerebellar Granule Cell Precursor Proliferation

We have previously shown that mice with a CNS restricted knock-out of the integrin β1 subunit gene (Itgb1-CNSko mice) have defects in the formation of lamina and folia in the cerebral and cerebellar cortices that are caused by disruption of the cortical marginal zones. Cortical structures in postnatal and adult Itgb1-CNSko animals are also reduced in size, but the mechanism that causes the size defect has remained unclear. We now demonstrate that proliferation of granule cell precursors (GCPs) is severely affected in the developing cerebellum of Itgb1-CNSko mice. In the absence of β1 expression, GCPs lose contact with laminin in the meningeal basement membrane, cease proliferating, and differentiate prematurely. In vitro studies provide evidence thatβ1 integrins act at least in part cell autonomously in GCPs to regulate their proliferation. Previous studies have shown that sonic hedgehog (Shh)-induced GCP proliferation is potentiated by the integrin ligand laminin. We show that Shh directly binds to laminin and that laminin–Shh induced cell proliferation is dependent on β1 integrin expression in GCPs. Taken together, these data are consistent with a model in which β1 integrin expression in GCPs is required to recruit a laminin–Shh complex to the surface of GCPs and to subsequently modulate the activity of signaling pathways that regulate proliferation.

β1 Integrins in Radial Glia But Not in Migrating Neurons Are Essential for the Formation of Cell Layers in the Cerebral Cortex

Radial glial cells in the cerebral cortex serve as progenitors for neurons and glia and guide the migration of cortical neurons. The integrin α3β1 is thought to mediate interactions of migrating neurons with radial glial cells and to function as a receptor for the reelin signaling molecule. Here, we challenge this view and demonstrate that β1 integrins in migrating neurons are not essential for the formation of cell layers in the cerebral cortex. Cortical cell layers also form normally in mice deficient in the integrin α3β1. However, we provide evidence that β1 integrins in radial glia control the morphological differentiation of both glia and neurons. We conclude that β1 integrins in radial glia are required for the proper development of the cerebral cortex, whereas β1 integrins in migrating neurons are not essential for glial-guided migration and reelin signaling.