ErbB2/neu kinase modulates cellular p27(Kip1) and cyclin D1 through multiple signaling pathways

It is well established that ErbB1 and ErbB2 can cooperate in mammary epithe lial cell transformation. Therefore, to understand how ErbB1/ErbB2 signalin g contributes to this process, we used the ErbB kinase inhibitor AG1478 in ErbB2-dependent BT-474 and SKBR-3 human breast cancer cells. These cells ov erexpress ErbB2 and also display moderate levels of ErbB1. Treatment with A G1478 resulted in rapid ErbB2 dephosphorylation, reversible G(1) arrest, an d interruption of constitutive mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Consequently, both MAPK -dependent transcription of cyclin DI and phosphorylation of the cyclin-dep endent kinase (Cdk) inhibitor p27 were inhibited. The inhibition of PI3K/Ak t resulted in increased activity of glycogen synthase kinase-3 beta, which phosphorylated cyclin D1, potentially reducing its steady-state levels. The loss of cyclin D1 reduced the amount of cyclin D1/Cdk4 complexes that can sequester p27 in the cytosol. This plus the reduced phosphorylation of p27 by MAPK enhanced the stability of p27 that associated with nuclear Cdk2 at high stoichiometry and inhibited its kinase activity. Antisense p27 oligonu cleotides decreased p27 levels and abrogated the G(1) arrest induced by AG1 478. Similarly, infection with an adenovirus encoding inducible cyclin D1 a lso counteracted the antiproliferative effect of AG1478. These data imply t hat: (a) modulation of both p27 and cyclin D1 are required for the growth a rrest that results from blockade of the ErbB2 kinase; and (b) ErbB2 overexp ressing cells use both MAPK and PI3K/Akt to modulate p27 and cyclin D1 and, hence, subvert the G(1)-to-S transition.