Julie A. Lofgren

Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth.

ErbB2 is a ligand-less member of the ErbB receptor family that functions as a coreceptor with EGFR, ErbB3, and ErbB4. Here, we describe an approach to target ErbB2s role as a coreceptor using a monoclonal antibody, 2C4, which sterically hinders ErbB2s recruitment into ErbB ligand complexes. Inhibition of ligand-dependent ErbB2 signaling by 2C4 occurs in both low- and high-ErbB2-expressing systems. Since the ErbB3 receptor contains an inactive tyrosine kinase domain, 2C4 is very effective in blocking heregulin-mediated ErbB3-ErbB2 signaling. We demonstrate that the in vitro and in vivo growth of several breast and prostate tumor models is inhibited by 2C4 treatment.

Heregulins and the ErbB-2/3/4 receptors in gliomas

The activation of autocrine loops involving proto-oncogene relatedreceptor tyrosine kinases has led to the analysisof a large number of growth factor systemsin human glioma specimens and cell lines. TheErbB-2 system, also called HER-2 or neu, isanalogous to the epidermal growth factor receptor system(EGF-R, ErbB-1). Neuregulins consist of a large familyof proteins arising from alternative mRNA splicing ofa single gene located at 8p22-p11. Activation ofErbB-2 by neuregulins occurs in heterodimeric complexes withErbB-3 and ErbB-4. A panel of human gliomacell lines, which had previously been analyzed forErbB-2 expression, was examined for ErbB-3 and ErbB-4expression. Coordinate expression of ErbB-2, -3 or -4was not observed in these cell lines. Despitethe presence of a complete system capable ofsignaling in about half the cell lines, noconstitutive activation of ErbB-2, -3 or -4 wasobserved, and autophosphorylation of ErbB-2 in response toheregulin was observed only in one cell linefrom the panel, NCE-G84. Moreover, the addition ofrecombinant heregulin or antibodies capable of disrupting ErbB-2/ErbB-3complexes had no effect on cell proliferation. Weconclude that the role of neuregulins and itsreceptors in the control of glioma cell proliferationmay be limited or may be context dependenton in situ conditions which are lost invitro. Alternatively, neuregulins may be involved in celldifferentiation or survival in the central nervous system.Data supporting these conclusions are described in moredetail herein.

Kinetic mechanism of AKT/PKB enzyme family.

AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.