Mechanisms linking mitogenic and growth inhibitory cytokine signaling and the cell cycle have not been fully elucidated in either cancer or in normal cells. Here we show that activation of protein kinase B (PKB)/Akt, contributes to resistance to antiproliferative signals and breast cancer progression in part by impairing the nuclear import and action of p27. Akt transfection caused cytoplasmic p27 accumulation and resistance to cytokine-mediated G1 arrest. The nuclear localization signal of p27 contains an Akt consensus site at threonine 157, and p27 phosphorylation by Akt impaired its nuclear import in vitro. Akt phosphorylated wild-type p27 but not p27T157A. In cells transfected with constitutively active Akt(T308DS473D)(PKB(DD)), p27WT mislocalized to the cytoplasm, but p27T157A was nuclear. In cells with activated Akt, p27WT failed to cause G1 arrest, while the antiproliferative effect of p27T157A was not impaired. Cytoplasmic p27 was seen in 41% (52 of 128) of primary human breast cancers in conjunction with Akt activation and was correlated with a poor patient prognosis. Thus, we show a novel mechanism whereby Akt impairs p27 function that is associated with an aggressive phenotype in human breast cancer.

We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTPmediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase. INTRODUCTIONThe Cdk inhibitor p27 is an important regulator of G1 progression. It is highly expressed in G0, where it binds tightly and inhibits cyclin E-Cdk 2 Polyak et al., 1994;Slingerland et al., 1994). In mid-G1, p27 also plays a role in the assembly and nuclear import of D-type cyclinCdk complexes (LaBaer et al., 1997;Cheng et al., 1999). p27 levels are regulated by translational controls and by proteolysis, and decrease as cells progress from G1 to S phase (Hengst and Reed, 1996;Millard et al., 1997;Slingerland and Pagano, 2000). The ubiquitin-dependent proteolysis of p27 (Pagano et al., 1995) is regulated by its phosphorylation at threonine 187 (T187) by cyclin E-Cdk 2 in late G1 and S phase (Sheaff et al., 1997;Vlach et al., 1997;Montagnoli et al., 1999). T187 phosphorylation allows recognition of p27 by its SCF-type E3 ligase, comprised of Skp1, Cul1, and the F-box protein, Skp2 and Roc1 and the Cks1 cofactor Ohta et al., 1999;Sutterluty et al., 1999;Tsvetkov et al., 1999;Ganoth et al., 2001;Spruck et al., 2001). Recent evidence suggests that p27 proteolysis is regulated by at least two distinct mechanisms, with mitogenic signaling conditioning p27 for degradation in early G1 in a manner independent of T187 phosphorylation (Hara et al., 2001;Malek et al., 2001), whereas Skp2-dependent cyclin E-Cdk 2-mediated degradation occurs in S phase after T187 phosphorylation (Malek et al., 2001). Although p27 is detected in the nuclei of most normal quiescent cells (Slingerland and Pagano, 2000), the relationship between its int...

Valproic acid (VPA) is a widely used antiepileptic agent that is undergoing clinical evaluation for anticancer therapy. We assessed the effects of VPA on angiogenesis in vitro and in vivo. In human umbilical vein endothelial cells, therapeutically relevant concentrations of VPA (0.25 to 1 mM) inhibited proliferation, migration, and tube formation. VPA 1 mM inhibited endothelial cell proliferation by 51 Ϯ 5%, migration by 86 Ϯ 11%, and tube formation by 82 Ϯ 3%. These changes were preceded by the hyperacetylation of histone H4, indicating the inhibition of histone deacetylase (HDAC), and a decreased expression of the endothelial nitric-oxide synthase (eNOS). The inhibition of endothelial cell tube formation by VPA was prevented by addition of the nitric oxide donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate). The anticonvulsive active VPA derivative 2-ethyl-4-methylpentanoic acid, which does not inhibit HDAC, did not affect endothelial cell proliferation, tube formation, or eNOS expression. VPA was also found to inhibit angiogenesis in vivo in the chicken chorioallantoic membrane assay and in a Matrigel plug assay in mice. Embryos from VPA-treated mice showed disturbed vessel formation. These results indicate that therapeutic plasma levels of VPA inhibit angiogenesis by a mechanism involving a decrease in eNOS expression preceded by HDAC inhibition.

A high frequency of human cytomegalovirus (HCMV) genome and antigens in tumor samples of patients with different malignancies is now well documented, although the causative role for HCMV in the development of the neoplasias remains to be established. HCMV infection can modulate multiple cellular regulatory and signalling pathways in a manner similar to that of oncoproteins of small DNA tumor viruses such as human papilloma virus or adenoviruses. However, in contrast to these DNA tumor viruses, HCMV infection fails to transform susceptible normal human cells. There is now growing evidence that tumor cells with disrupted regulatory and signalling pathways enable HCMV to modulate their properties including stimulation of cell proliferation, survival, invasion, production of angiogenic factors, and immunogenic properties. In contrast to previously suggested "hit and run" transformation we suggest that persistence in tumor cells is essential for HCMV to fully express its oncomodulatory effects. These effects are observed particularly in persistent HCMV infection and are mediated mainly by activity of HCMV regulatory proteins. In persistently HCMV-infected tumor cell lines - a selection of novel, slowly growing virus variants with changes in coding sequences for virus regulatory proteins takes place. As a result, oncomodulatory effects of HCMV infection may lead to a shift to more malignant phenotype of tumor cells contributing to tumor progression.