Forkhead transcription factors FOXO1 (FKHR), FOXO3a (FKHRL1), and FOXO4 (AFX) play a pivotal role in tumor suppression by inducing growth arrest and apoptosis. Loss of function of these factors due to phosphorylation and proteasomal degradation has been implicated in cell transformation and malignancy. However, the ubiquitin ligase necessary for the ubiquitination of the FOXO factors and the relevance of this regulation to tumorigenesis have not been characterized. Here we demonstrate that Skp2, an oncogenic subunit of the Skp1͞Cul1͞F-box protein ubiquitin complex, interacts with, ubiquitinates, and promotes the degradation of FOXO1. This effect of Skp2 requires Akt-specific phosphorylation of FOXO1 at Ser-256. Moreover, expression of Skp2 inhibits transactivation of FOXO1 and abolishes the inhibitory effect of FOXO1 on cell proliferation and survival. Furthermore, expression of the FOXO1 protein is lost in a mouse lymphoma model, where Skp2 is overexpressed. These data suggest that the Skp2-promoted proteolysis of FOXO1 plays a key role in tumorigenesis.ubiquitin ligase ͉ proteasomal degradation ͉ cancer F orkhead family members FOXO1, FOXO3a, and FOXO4 are multifunctional transcription factors, which regulate transcription of a number of genes that play critical roles in inducing either cell cycle arrest or apoptosis. Activation of each member of this family in transformed and nontransformed cells results in upregulation of the cyclin-dependent kinase inhibitor p27 KIP1 and͞or down-regulation of D-type cyclins, thereby arresting cells at G 1 (1, 2). Activated FOXO proteins also trigger apoptosis in many cancer cell lines through regulation of a number of proapoptotic proteins, including Fas ligand, TRAIL, and Bim (3-5). Knocking down the FOXO3a protein in human breast cancer cells or inhibition of the transcriptional activity of FOXO1 in chicken embryo fibroblasts promotes cell transformation and tumor progression (6, 7). Thus, it has been postulated that FOXO factors play a pivotal role in the inhibition of cell transformation and tumorigenesis.The inhibitory function of FOXO proteins in cell proliferation and survival is often disrupted due to the overactivated phosphatidylinositol 3-kinase (PI3K)͞Akt pathway in cancer cells. Activated Akt phosphorylates a wide range of downstream proapoptotic proteins, among which are the forkhead factors FOXO1, FOXO3a, and FOXO4. Phosphorylated forkhead proteins translocate from the nucleus to the cytoplasm where they are inactive (3,(8)(9)(10). Recently, another kinase, I B kinase ␤, has been shown to phosphorylate and inactivate FOXO3a in breast cancer cells (6). The tumor suppressor gene PTEN encodes a lipid phosphatase that specifically dephosphorylates the D3 position of phosphatidylinositol 3,4,5-trisphosphate (11) and in so doing functionally antagonizes the PI3K pathway. Because of frequent deletions and mutations in the PTEN gene in human cancers, it is believed that protein phosphorylation is a key mechanism that inactivates the FOXO factors.It has been demonstra...

The function of cyclin-dependent kinase 2 (CDK2) is often abolished after DNA damage. The inhibition of CDK2 plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences the survival of cells under genotoxic stress is unknown. Forkhead box O (FOXO) transcription factors are emerging as key regulators of cell survival. CDK2 specifically phosphorylated FOXO1 at serine-249 (Ser249) in vitro and in vivo. Phosphorylation of Ser249 resulted in cytoplasmic localization and inhibition of FOXO1. This phosphorylation was abrogated upon DNA damage through the cell cycle checkpoint pathway that is dependent on the protein kinases Chk1 and Chk2. Moreover, silencing of FOXO1 by small interfering RNA diminished DNA damage-induced death in both p53-deficient and p53-proficient cells. This effect was reversed by restored expression of FOXO1 in a manner depending on phosphorylation of Ser249. Functional interaction between CDK2 and FOXO1 provides a mechanism that regulates apoptotic cell death after DNA strand breakage.

A Francisella tularensis live vaccine strain mutant (sodB Ft ) with reduced Fe-superoxide dismutase gene expression was generated and found to exhibit decreased sodB activity and increased sensitivity to redox cycling compounds compared to wild-type bacteria. The sodB Ft mutant also was significantly attenuated for virulence in mice. Thus, this study has identified sodB as an important F. tularensis virulence factor.

Systemic prostate cancer therapy requires androgen ablation, which inhibits the production or action of androgens. Prostate cancer ultimately relapses during androgen ablation, and an androgen depletion-independent (ADI) phenotype emerges. Aberrant androgen receptor (AR) activation underlies therapy resistance at this stage of the disease, and mounting evidence implicates the large and highly disordered AR NH 2 -terminal domain (NTD) as a key mediator of this activity. In this study, we investigated the role of the NTD transactivation unit 5 (TAU5) domain in mediating AR transcriptional activity in cell-based models of prostate cancer progression. AR replacement and Gal4-based promoter tethering experiments revealed that AR TAU5 had a dichotomous function, inhibiting ligand-dependent AR activity in androgen-dependent prostate cancer cells, while enhancing ligand-independent AR activity in ADI prostate cancer cells. Molecular dissection of TAU5 showed that a WxxLF motif was fully responsible for its ligandindependent activity. Mechanistically, WxxLF did not rely on an interaction with the AR ligand-binding domain to mediate ligand-independent AR activity. Rather, WxxLF functioned as an autonomous transactivation domain. These data show that ligand-dependent and ligand-independent AR activation rely on fundamentally distinct mechanisms, and define WxxLF as the major transactivation motif within the AR TAU5 domain.

Standard therapy for nonorgan confined prostate cancer aims to block the production or action of androgens. Although initially successful, antiandrogen therapy eventually fails and androgen depletion independent (ADI) disease emerges. Remarkably, ADI prostate cancers still rely on a functional androgen receptor (AR). Aberrant expression of coregulatory proteins required for the formation of productive AR transcriptional complexes is critical for ADI AR activation. Previously, we have shown that the transcriptional coactivator p300 is required for ADI activation of the AR and is upregulated in prostate cancer, in which its expression is associated with cell proliferation and predicts aggressive tumor features. The mechanism responsible for the deregulated expression of p300, however, remains elusive. Here, we show that p300 expression in prostate cancer cells is subject to androgen regulation. In several prostate cancer model systems, addition of synthetic and natural androgens led to decreased expression of p300 in a time-dependent and dose-dependent manner. Experiments using AR antagonists or small interfering RNA targeting the AR revealed that down-regulation of p300 depends entirely on the presence of a functional AR. It is noteworthy that androgens down-regulated p300 protein expression while leaving messenger levels unaltered. Conversely, both short-term and long-term androgen deprivation resulted in marked up-regulation of p300 expression. The androgen deprivation-induced increase in p300 expression was not affected by the addition of cytokines or growth factors or by cotreatment with antiandrogens. Moreover, increased p300 expression upon androgen starvation is crucial for prostate cancer cell proliferation, as loss of p300 expression severely reduces expression of cyclins governing G 1 -S and G 2 -M cell cycle transition and decreases 5-bromo-2 ¶-deoxyuridine incorporation. [Cancer Res 2007;67(7):3422-30]