We have shown a novel mechanism of Akt-mediated regulation of the CDK inhibitor p27(kip1). Blockade of HER2/neu in tumor cells inhibits Akt kinase activity and upregulates nuclear levels of the CDK inhibitor (Kip1). Recombinant Akt and Akt precipitated from tumor cells phosphorylated wild-type p27 in vitro. p27 contains an Akt consensus RXRXXT(157)D within its nuclear localization motif. Active (myristoylated) Akt phosphorylated wild-type p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wild-type p27 localized in the cytosol and nucleus, whereas T157A-p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by Akt. T157A-p27 was more effective than wild-type p27 in inhibiting cyclin E/CDK2 activity and cell proliferation; these effects were not rescued by active Akt. Expression of Ser(473) phospho Akt in primary human breast cancers statistically correlated with expression of p27 in tumor cytosol. These data indicate that Akt may contribute to tumor-cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.

We have examined the possible mechanisms of resistance to the epidermal growth factor receptor (EGFR) inhibitors in tumor cells with variable levels of EGFR. ZD1839 (Iressa) is a small-molecular-weight, ATP-mimetic that specifically inhibits the EGFR tyrosine kinase. A431 cell growth was markedly inhibited by ZD1839 (IC 50 p0.1 lm) whereas the MDA-468 cells were relatively resistant (IC 50 >2 lm). Low doses of ZD1839 delayed cell cycle progression and induced apoptosis in A431 cells but not in MDA-468 cells. In both cell lines, 0.1 lm ZD1839 eliminated EGFR phosphorylation. However, the basal activity of the phosphatidylinositol-3 kinase (PI3 K) target Akt was eliminated in A431 but not in MDA-468 cells, implying that their Akt activity is independent of EGFR signals. A431 cells express PTEN/MMAC1/TEP, a phosphatase that can dephosphorylate position D3 of phosphatidylinositol-3,4,5 trisphosphate, the site that recruits the plecstrin-homology domain of Akt to the cell membrane. On the contrary, MDA-468 cells lack the phosphatase and tensin homolog (PTEN), potentially setting Akt activity at a high threshold that is unresponsive to EGFR inhibition alone. Therefore, we reintroduced (PTEN) by retroviral infection in MDA-468 cells. In MDA-468/PTEN but not in vector controls, treatment with ZD1839 inhibited P-Akt levels, induced relocalization of the Forkhead factor FKHRL1 to the cell nucleus, and increased FKHRL1-dependent transcriptional activity. ZD1839 induced a greater degree of apoptosis and cell cycle delay in PTEN-reconstituted than in control cells. These data suggest that loss of PTEN, by permitting a high level of Akt activity independent of receptor tyrosine kinase inputs, can temporally dissociate the inhibition of the EGFR with that of Akt induced by EGFR inhibitors. Thus, in EGFR-expressing tumor cells with concomitant amplification(s) of PI3K-Akt signaling, combined blockade of the EGFR tyrosine kinase and Akt should be considered as a therapeutic approach.

We have examined the interaction of transforming growth factor (TGF)␤ receptors with phosphatidylinositol 3-(PI3) kinase in epithelial cells. In COS7 cells, treatment with TGF␤ increased PI3 kinase activity as measured by the ability of p85-associated immune complexes to phosphorylate inositides in vitro. Both type I and type II TGF␤ receptors (T␤R) associated with p85, but the association of T␤RII appeared to be constitutive. The interaction of T␤RI with p85 was induced by treatment with TGF␤. The receptor association with PI3 kinase was not direct as 35 S-labeled rabbit reticulocyte p85 did not couple with fusion proteins containing type I and type II receptors. A kinase-dead, dominant-negative mutant of T␤RII blocked ligand-induced p85-⌻␤RI association and PI3 kinase activity. In T␤RI-null R1B cells, TGF␤ did not stimulate PI3 kinase activity. This stimulation was restored upon reconstitution of T␤RI by transfection. In R1B and NMuMG epithelial cells, overexpression of a dominant active mutant form of T␤RI markedly enhanced ligand-independent PI3 kinase activity, which was blocked by the addition of the T␤RI kinase inhibitor LY580276, suggesting a causal link between T␤RI function and PI3 kinase. Overexpressed Smad7 also prevented ligand-induced PI3 kinase activity. Taken together, these data suggest that 1) TGF␤ receptors can indirectly associate with p85, 2) both receptors are required for ligand-induced PI3 kinase activation, and 3) the activated T␤RI serine-threonine kinase can potently induce PI3 kinase activity.Transforming growth factor ␤ (TGF␤) 1 binds to a heteromeric complex of transmembrane serine-threonine kinases, the type I and II TGF␤ receptors (T␤RI or Alk5 and T␤RII). Following ligand binding to T␤RII, the type I receptor is recruited to the ligand-receptor complex where the constitutively active T␤RII transactivates T␤RI (1). Activated T␤RI phosphorylates the receptor-specific Smad2 and Smad3, which then associate with Smad4 and, as a heteromeric complex, translocate to the nucleus where they regulate the transcription of TGF␤ target genes (1). The Smad signaling pathway mediates the antiproliferative effect of TGF␤ in epithelial cells (2, 3) and is the best characterized. Several non-Smad pathways have also been implicated in mediating the cellular effects of TGF␤. These include the extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 (PI3) kinase, and the family of Rho GTPases (4, 5).Activated PI3 kinase increases the formation of intracellular 3Ј-phosphorylated inositol lipids, signal transducers that are essential for the regulation of cell cycle progression, glucose metabolism, cell motility, epithelial-to-mesenchymal transition, and apoptosis among others (6, 7). In Swiss 3T3 cells, TGF␤ stimulates PI3 kinase activity, as measured by the ability of immune complexes precipitated with an antibody against p85, the regulatory subunit of PI3 kinase, to induce the formation of phosphatidylinositol-3 mo...

The Forkhead family of transcription factors participates in the induction of death-related genes. In NMuMG and 4T1 mammary epithelial cells, transforming growth factor beta (TGF beta) induced phosphorylation and cytoplasmic retention of the Forkhead factor FKHRL1, while reducing FHKRL1-dependent transcriptional activity. TGF beta-induced FKHRL1 phosphorylation and nuclear exclusion were inhibited by LY294002, an inhibitor of phosphatidylinositol-3 kinase. A triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated (TM-FKHRL1), did not translocate to the cytoplasm in response to TGF beta. In HaCaT keratinocytes, expression of dominant-negative Akt prevented TGF beta-induced 1) reduction of Forkhead-dependent transcription, 2) FKHRL1 phosphorylation, and 3) nuclear exclusion of FKRHL1. Forced expression of either wild-type (WT) or TM-FKHRL1, but not a FKHRL1 mutant with deletion of the transactivation domain, resulted in NMuMG mammary cell apoptosis. Evidence of nuclear fragmentation colocalized to cells with expression of WT- or TM-FKHRL1. The apoptotic effect of WT-FKHRL1 but not TM-FKHRL1 was prevented by exogenous TGF beta. Serum starvation-induced apoptosis was also inhibited by TGF beta in NMuMG and HaCaT cells. Finally, dominant-negative Akt abrogated the antiapoptotic effect of TGF beta. Taken together, these data suggest that TGF beta may play a role in epithelial cell survival via Akt-dependent regulation of FKHRL1.

We generated a p27Kip1 mutant (p27#NLS) that localized exclusively in cell cytosol. Expression of p27#NLS in MCF7 breast cancer cells down-regulated RhoA and increased motility, survival, and Akt levels without an effect on cell cycle distribution. RNA interference of p27 in U87 glioma cells, which express p27 predominantly in the cytoplasm, inhibited motility and survival. Conversely, knockdown of p27 in COS7 cells, with >95% nuclear p27 expression, accelerated proliferation but had no effect on motility or survival. U87 cells in which p27 had been eliminated by RNA interference exhibited lower Akt levels, shorter Akt turnover, and markedly impaired tumorigenicity in vivo. These xenografts were less invasive and exhibited increased apoptosis compared with p27-expressing tumors. Expression of cytosolic p27 in primary human breast carcinomas correlated linearly with Akt content as measured by immunohistochemistry. These data suggest that cytoplasmic p27 can exert oncogenic functions by modulating Akt stability, cell survival, and tumorigenicity. (Cancer Res 2006; 66(4): 2162-72)