Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu + MMTV/TGF-α bigenic mice

Abstract

Overexpression of ErbB-2͞Neu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)͞Neu ؉ MMTV͞transforming growth factor ␣ bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogenactivated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27 Kip1 . In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly in vitro by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1͞2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 and MAPK precipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant in vitro. Cdk2 and MAPK precipitates from AG-1478-treated tumors were unable to phosphorylate p27 in vitro. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression.T he ErbB family of receptors includes the epidermal growth factor receptor (EGFR) ErbB-1, the orphan ErbB-2 (Neu), and the Neuregulin receptors ErbB-3 and ErbB-4 (1-4). Binding of ligands to the ectodomain of these receptors results in the formation of homodimeric and heterodimeric complexes (5), which is followed rapidly by the activation of the receptors' intrinsic tyrosine kinase. Consequently, phosphorylation of specific C-terminal tyrosine residues and the recruitment of specific second messengers activate a plethora of intracellular signaling pathways that play central roles in cell proliferation, development, differentiation, migration, and oncogenesis (6).Many studies support a pivotal role for the orphan ErbB-2͞ Neu in ErbB signaling and tumorigenesis (7-9), suggesting that this receptor plays a critical role in the cellular responses mediated by the ligand-dependent activation of other ErbB receptors (10, 11). Indeed, overexpression of ErbB-2͞Neu alone or in combination with EGFR (ErbB-1) or ErbB-3 in vitro can transform mammary epithelial cells (8) and fibroblasts (12, 13), respectively. A central role for ErbB-2͞Neu in transformation has been shown by using transgenic mice overexpressing the protooncogene under the control of the mouse mammary tumor virus (MMTV) promoter (14, 15). In these tumors, DNA sequence analysis revealed the presence of a 16-aa in-frame deletion in the extracellular domain of ErbB-2͞Neu, resulting in a constitutively activated receptor capable of transforming Rat-1 fibroblasts (16). So far, no such activating mutations have been found in human tumors, where the m...

The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition

“…The levels of p27 are high in quiescent cells and decline in response to mitogenic factor stimulation. Several studies have suggested that the Ras-ERK1/2 signaling pathway is involved in the mitogen-induced downregulation of p27 (Kawada et al, 1997;Kerkhoff and Rapp, 1997;Woods et al, 1997;Greulich and Erikson, 1998;Rivard et al, 1999;Treinies et al, 1999;Lenferink et al, 2000;Yang et al, 2000;Delmas et al, 2001;Mirza et al, 2004;Gysin et al, 2005;Sakakibara et al, 2005). In contrast, other studies failed to document any significant change in p27 levels following inhibition of ERK1/2 signaling by synthetic MEK1/2 inhibitors or dominant-negative ERK2, or after conditional activation of the pathway by activated Raf-1 or MEK1 (Sewing et al, 1997;Takuwa and Takuwa, 1997;Weber et al, 1997a;Cheng et al, 1998;Ladha et al, 1998;Chen et al, 1999;Tetsu and McCormick, 2003).…”

Early immune responses are independent of RGC dysfunction in glaucoma with complement component C3 being protective

“…The dose delivered by the pump was 12 mg·kg −1 ·d −1 for an average of 30 g per mouse. AG1478 has a short half-life (<60 min) in vivo (43,44), but has been shown to successfully inhibit EGFR activity with continuous infusion at similar concentrations (45). Twenty-five mice were treated with AG1487 and 25 mice were treated with vehicle, although a few mice were lost due to normal age-related attrition.…”

TGFβ prevents proteasomal degradation of the cyclin-dependent kinase inhibitor p27kip1 for cell cycle arrest

“…www.landesbioscience.com Cell Cycle 749 cancers, 72,73 phosphorylation of p27 by these kinases would likely lead to elevated levels of Cdk2. p27 in the cytoplasm has been shown to colocalize with actin and mediate cellular migration and metastasis by blocking Rho stress fiber formation.…”