Transforming growth factor-beta (TGF-beta) mediates cell cycle arrest by activating cdk inhibitors, principle among which is p27[Kip1]. Loss of growth inhibition by TGF-beta occurs early in oncogenesis and contributes to tumor progression. We have studied cell cycle effects of TGF-beta and how they are lost in human tumors, p27 is often inactivated in human cancers due to accelerated p27 proteolysis or cytoplasmic mislocalization of p27. In cancer cells, progressive checkpoint loss makes p27 essential for TGF-beta mediated arrest. We showed activation of the phosphoinositol 3' kinase (PI3K) effector, protein kinase B (PKB) phosphorylates p27 at threonine 157, mislocalizating p27 in the cytoplasm. PKB activated cells also showed increased p27 in cyclin D1-cdk complexes. Preliminary data suggest that other effectors of the PI3K pathway may also inhibit p27 function. PI3K and its downstream effector pathways are often activated in cancers by oncogenic receptor tyrosine kinase overexpression, activating ras mutations or loss of the PI3K inhibitor, PTEN. Here we investigate how p27 function and G1 arrest by TGF-beta may be opposed by mitogenic PI3K signaling in cancers. Our hypothesis is that constitutive activation of the PI3K pathway leads to p27 phosphorylation events that reduce its affinity for cyclin E-cdk2, alter its stability and intraceltular localization, and abrogate its inhibitory function leading to TGF-beta resistance. This is pursued by the following specific aims:
AIM 1) to assay if PKB activates p27 assembly function toward cyclin D1-cdk4 and reduces p27's inhibitory action toward cyclin E-cdk2;
AIM 2) to determine whether PI3K effectors other than PKB can mediate TGF-beta resistance;
AIM 3) to assay if other PI3K dependent kinases can phosphorylate p27 and alter its function and AIM 4) to investigate whether PI3K effectors impair nuclear import of p27. These studies may link TGF-beta resistance to oncogenic activation of mitogenic P13K signaling pathways commonly observed in human cancers. Elucidation of molecular mechanisms of p27 inactivation and TGF-beta resistance in cancers may yield new targets for therapeutic intervention.
