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* This work was supported in part by National Institutes of Health Grants CA70297 and CA87767 and the Department of Army Prostate Cancer Grant PC01-0690.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
PI3K/Akt plays a critical role in prostate cancer cell growth and survival. Recent studies have shown that the effect of PI3K/Akt in prostate cells is mediated through androgen signaling. The PI3K inhibitor, LY294002, and a tumor suppressor, PTEN, negatively regulate the PI3K/Akt pathway and repress AR activity. However, the molecular mechanisms whereby PI3K/Akt and PTEN regulate the androgen pathway are currently unclear. Here, we demonstrate that blocking the PI3K/Akt pathway reduces the expression of an endogenous AR target gene. Moreover, we show that the repression of AR activity by LY294002 is mediated through phosphorylation and inactivation of GSK3β, a downstream substrate of PI3K/Akt, which results in the nuclear accumulation of β-catenin. Given the recent evidence that β-catenin acts as a coactivator of AR, our findings suggest a novel mechanism by which PI3K/Akt modulates androgen signaling. In a PTEN-null prostate cancer cell line, we show that PTEN expression reduces β-catenin-mediated augmentation of AR transactivation. Using the mutants of β-catenin, we further demonstrate that the repressive effect of PTEN is mediated by a GSK3β-regulated degradation of β-catenin. Our results delineate a novel link among the PI3K, wnt, and androgen pathways and provide fresh insights into the mechanisms of prostate tumor development and progression.
glycogen synthase kinase 3β
phosphatase and tensin homolog deleted on chromosome 10
Prostate cancer is the most common malignancy in men and the second leading cause of cancer death in the United States (
). The growth-promoting effects of androgen in prostate cells are mediated mostly through the androgen receptor (AR).1 The AR belongs to the nuclear receptor superfamily and acts as a ligand-dependent transcription factor (
The phosphatidylinositol 3-kinase (PI3K) consists of regulatory (p85) and catalytic (p110) subunits that participate in multiple cellular processes including cell growth, transformation, differentiation, and survival (
). The binding of PI3K-generated phospholipids to Akt results in the translocation of Akt from the cytoplasm to the inner surface of the plasma membrane where Akt is phosphorylated by the upstream kinases, PDK-1, PDK-2, and ILK (
). The activation of Akt results in the phosphorylation of a number of downstream substrates such as glycogen synthase kinase (GSK3), Bad, and caspase9 and the forkhead transcription factors, Raf, Iκb kinase, and phosphodiesterase 3B (
). As one of the principal physiological substrates of Akt, GSK3 is a ubiquitously expressed protein serine/threonine kinase that was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (
). Corresponding to its dual functions in cells, β-catenin is localized to two cellular pools. Most of the β-catenin is located in the cell membrane where it is associated with the cytoplasmic region of E-cadherin, a transmembrane protein involved in homotypic cell-cell contacts (
). A smaller pool of β-catenin is located in both the nucleus and cytoplasm where it mediates Wnt signaling. In the absence of a Wnt signal, β-catenin is constitutively down-regulated by a multicomponent destruction complex containing GSK3β, axin, and the tumor suppressor adenomatous polyposis coli. These proteins promote the phosphorylation of serine and threonine residues in the amino-terminal region of β-catenin and thereby target it for degradation by the ubiquitin proteasome pathway (
). Although several potential mechanisms have been suggested for this cross-talk, the precise molecular basis by which PI3K/AKT and PTEN regulate AR-mediated transcription is currently unclear. Recently, a specific protein-protein interaction between β-catenin and AR was identified by us and others (
). Through this interaction, β-catenin augments the ligand-dependent activity of AR in prostate cancer cells. Here, we provide multiple lines of evidence showing that the cross-talk between the androgen and PI3K/Akt pathways is mediated through the modulation of the PI3K/Akt downstream effector GSK3β. Its inactivation by phosphorylation results in increased nuclear levels of β-catenin, which augment AR activity. These findings delineate a novel mechanism by which PI3K/Akt and PTEN regulate the androgen pathway during prostate cell growth and survival.
The PI3/Akt pathway plays a critical role in prostate cell proliferation and survival (
). PTEN, which is frequently mutated in prostate cancer cells, negatively regulates this process by blocking the PI3K/Akt pathway. Recently, several lines of evidence showed that PI3K/Akt and PTEN can modulate androgen-induced cell growth and AR-mediated transcription in prostate cancer cells (
), suggesting a potential link between the PI3K/Akt and androgen pathways. In this study, we demonstrated that β-catenin acts as the point of convergence for the cross-talk between the PI3K/Akt and androgen signaling pathways. The data presented here are consistent with what is known regarding the degradation of β-catenin by GSK3β, a downstream effector of PI3K/Akt, and fit very well with our recent finding that β-catenin interacts with AR and augments its ligand-dependent transcription (
). Tumor cells contain high levels of free cellular β-catenin by acquiring loss-of-function mutations in the components of the destruction complex or by altering regulatory sequences in β-catenin itself. Besides Wnt signaling, other signaling pathways are also involved in regulating cellular β-catenin levels (
). In this study, we showed that PI3K/Akt increases the stability of nuclear β-catenin by phosphorylation and inactivation of the downstream substrate GSK3β in prostate cancer cells. Given that β-catenin acts as a transcriptional coactivator of AR, these data provide evidence to suggest a new mechanism whereby PI3K/Akt can affect prostate cell proliferation and survival through androgen signaling.
Earlier studies showed that PTEN negatively regulates the PI3K/Akt pathway in prostate cancer cells (
). The expression of PTEN in LNCaP, a PTEN-null prostate cancer cell line, blocks androgen-induced cell growth and AR-mediated transcription. In this study, we demonstrated that the overexpression of PTEN in LNCaP reduces β-catenin-mediated augmentation of AR activity; however, PTEN showed no effect in cells transfected with β-catenin mutants containing a single point mutation within the GSK3β phosphorylation sites. The results from our biochemical experiments further demonstrated that PTEN reduces the nuclear accumulation of β-catenin proteins in prostate cells. Because the β-catenin mutants used in our experiments are impervious to degradation by the destruction complex, we conclude that the regulation of β-catenin by PTEN is mediated through GSK3β. Our results are consistent with a recent study showing that nuclear β-catenin protein is constitutively elevated in PTEN null cells, and this elevated expression can be reduced upon the reexpression of PTEN (
). The data presented here also confirm that PTEN negatively regulates the PI3K pathway by inhibiting phosphorylation of Akt. In addition, the experiments using PTEN as a natural PI3K inhibitor are consistent with our data showing the important effects mediated by the synthetic PI3K inhibitor LY294002.
Modification of the AR protein such as by phosphorylation or acetylation has been suggested to be an important mechanism for modulating AR activity in prostate cancer cells (
). However, using both biochemical and functional approaches, we were not able to show a physical protein-protein interaction between Akt and AR or the phosphorylation of AR by Akt in vitro (data not shown). Results similar to ours were also reported by Li et al. (
). These conflicting results may be attributed to the use of different reagents and experimental conditions, but they also suggest that other alternative pathways may be involved in this regulation (Fig. 6). As presented in this study, we propose a novel molecular mechanism for PI3K/Akt and PTEN regulation of androgen signaling in prostate cancer cells.
The major role of β-catenin in tumorigenesis has been implicated via its interaction with the Tcf/LEF transcription factors (
), β-catenin is shown to have no effect on the activation of Tcf/LEF-mediated transcription in prostate cancer cells despite the expression of Tcf/LEF. A similar observation was also reported recently in breast cancer cells (
). In this study, using Tcf/LEF reporters, we were also not able to demonstrate an effect of PTEN on the regulation by β-catenin of Tcf/LEF-mediated transcription in LNCaP cells (data not shown). This raises the question as to whether the growth-promoting effect of β-catenin is mediated through partners outside of the Tcf/LEF pathway in prostate cancer and/or other tumor cells.
In this study, we demonstrate that β-catenin mediates the cross-talk between PI3K/Akt and androgen pathways. Based on these results and previous studies by others, we summarize our findings in Fig. 6. The PI3K/Akt signal induces phosphorylation and inactivation of GSK3β, resulting in increased nuclear levels of β-catenin. Consequently, increased β-catenin elevates AR activity to stimulate prostate cell growth and survival. Both the PI3K inhibitor LY294002 and PTEN negatively regulate these processes. A loss-of-expression or mutational inactivation of PTEN has been frequently observed in human tumors, which induce the suppression of apoptosis and accelerates cell cycle progression (
). Additionally, the mutation or aberrant expression of the destruction complex and the reduction of E-cadherin, which results in increased nuclear β-catenin, also occurs during prostate cancer progression (
). Our data showing that PTEN reduces nuclear β-catenin in prostate cancer cells suggest a novel role of PTEN in down-regulating androgen-induced cell growth and survival. A further study of the regulation of the interaction among PI3K, Wnt, and the androgen signaling pathways in prostate cancer cells should provide fresh insight into the pathogenesis of prostate cancer that may help us to identify new pathways that can be targeted for prostate cancer treatment.
We are especially grateful to Drs. Jan Trapman, Richard Roth, and William Sellers for the various reagents. We thank Homer Abaya for administrative assistance and help in preparing this paper.