PTEN: life as a tumor suppressor

PTEN, a tumor suppressor located at chromosome 10q23, is mutated in a variety of sporadic cancers and in two autosomal dominant hamartoma syndromes. PTEN is a phosphatase which dephosphorylates phosphatidylinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3), an important intracellular second messenger, lowering its level within the cell. By dephosphorylating PtdIns-3,4,5-P3, PTEN acts in opposition to phosphatidylinositol 3-kinase (PI3K), which has a pivotal role in the creation of PtdIns-3,4,5-P3. PtdIns-3,4,5-P3 is necessary for the activation of Akt, a serine/threonine kinase involved in cell growth and survival. By blocking the activation of Akt, PTEN regulates cellular processes such as cell cycling, translation, and apoptosis. In this review, we will discuss the identification of PTEN, its mutational status in cancer, its role as a regulator of PI3K, and its domain structure.     

Regulation of the activity of the tumor suppressor PTEN by thioredoxin in Drosophila melanogaster

Human Thioredoxin-1 (hTrx-1) is a small redox protein with a molecular weight of 12 kDa that contains two cysteine residues found in its catalytic site. HTrx-1 plays an important role in cell growth, apoptosis, and cancer patient prognosis. Recently, we have demonstrated that hTrx-1 binds to the C2 domain of the human tumor suppressor, PTEN, in a redox dependent manner. This binding leads to the inhibition of PTEN lipid phosphatase activity in mammalian tissue culture systems. In this study, we show that over-expression of hTrx-1 in Drosophila melanogaster promotes cell growth and proliferation during eye development as measured by eye size and ommatidia size. Furthermore, hTrx-1 rescues the small eye phenotype induced by the over-expression of PTEN. We demonstrate that this rescue of the PTEN-induced eye size phenotype requires cysteine-218 in the C2 domain of PTEN. We also show that hTrx-1 over-expression results in increased Akt phosphorylation in fly head extracts supporting our observations that the hTrx-1-induced eye size increase results from the inhibition of PTEN activity. Our study confirms the redox regulation of PTEN through disulfide bond formation with the hTrx-1 in Drosophila and suggests conserved mechanisms for thioredoxins and their interactions with the phosphatidylinositol-3-kinase signaling pathway in humans and fruit flies.     

Role of PTEN and Akt in the regulation of growth and apoptosis in human osteoblastic cells

Cancer cells are characterized by either an increased ability to proliferate or a diminished capacity to undergo programmed cell death. PTEN is instrumental in regulating the balance between growth and death in several cell types and has been described as a tumor suppressor. The chromosome arm on which PTEN is located is deleted in a subset of human osteosarcoma tumors. Therefore, we predicted that the loss of PTEN expression was contributing to increased Akt activation and the subsequent growth and survival of osteosarcoma tumor cells. Immunoblot analyses of several human osteosarcoma cell lines and normal osteoblasts revealed relatively abundant levels of PTEN. Furthermore, stimulation of cell growth or induction of apoptosis in osteosarcoma cells failed to affect PTEN expression or activity. Therefore, routine regulation of osteosarcoma cell growth and survival appears to be independent of changes in PTEN. Subsequently, the activation of a downstream target of PTEN activity, the survival factor Akt, was analyzed. Inappropriate activation of Akt could bypass the negative regulation by PTEN. Analyses of Akt expression in several osteosarcoma cell lines and normal osteoblasts revealed uniformly low basal levels of phosphorylated Akt. The levels of phosphorylated Akt did not increase following growth stimulation. In addition, osteosarcoma cell growth was unaffected by inhibitors of phosphatidylinositol-3 kinase, an upstream activator of the Akt signaling pathway. These data further suggest that the Akt pathway is not the predominant signaling cascade required for osteoblastic growth. However, inhibition of PTEN activity resulted in increased levels of Akt phosphorylation and enhanced cell proliferation. These data suggest that while abundant levels of PTEN normally maintain Akt in an inactive form in osteoblastic cells, the Akt signaling pathway is intact and functional.     

PTEN regulates RANKL- and osteopontin-stimulated signal transduction during osteoclast differentiation and cell motility

PTEN (also known as MMAC-1 or TEP-1) is a frequently mutated tumor suppressor gene in human cancer. PTEN functions have been identified in the regulation of cell survival, growth, adhesion, migration, and invasiveness. Here, we characterize the diverse signaling networks modulated by PTEN in osteoclast precursors stimulated by RANKL and osteopontin (OPN). RANKL dose-dependently stimulated transient activation of Akt before activation of PTEN, consistent with a role for PTEN in decreasing Akt activity. PTEN overexpression blocked RANKL-activated Akt stimulated survival and osteopontin-stimulated cell migration while a dominant-negative PTEN increased the actions of RANKL and OPN. PTEN overexpression suppressed RANKL-mediated osteoclast differentiation and OPN-stimulated cell migration. The PTEN dominant-negative constitutively induced osteoclast differentiation and cell migration. Our data demonstrate multiple roles for PTEN in RANKL-induced osteoclast differentiation and OPN-stimulated cell migration in RAW 264.7 osteoclast precursors.     

AIF inhibits tumor metastasis by protecting PTEN from oxidation

Apoptosis-inducing factor (AIF) exerts dual roles on cell death and survival, but its substrates as a putative oxidoreductase and roles in tumorigenesis remain elusive. Here, we report that AIF physically interacts with and inhibits the oxidation of phosphatase and tensin homolog on chromosome ten (PTEN), a tumor suppressor susceptible for oxidation-mediated inactivation. More intriguingly, we also identify PTEN as a mitochondrial protein and the ectopic expression of mitochondrial targeting sequence-carrying PTEN almost completely inhibits Akt phosphorylation in PTEN-deficient cells. AIF knockdown causes oxidation-mediated inactivation of the lipid phosphatase activity of PTEN, with ensuing activation of Akt kinase, phosphorylation of the Akt substrate GSK-3β, and activation of β-catenin signaling in cancer cells. Through its effect on β-catenin signaling, AIF inhibits epithelial–mesenchymal transition (EMT) and metastasis of cancer cells in vitro and in orthotopically implanted xenografts. Accordingly, the expression of AIF is correlated with the survival of human patients with cancers of multiple origins. These results identify PTEN as the substrate of AIF oxidoreductase and reveal a novel function for AIF in controlling tumor metastasis.     

PTEN expression causes feedback upregulation of insulin receptor substrate 2

PTEN is a tumor suppressor that antagonizes phosphatidylinositol-3 kinase (PI3K) by dephosphorylating the D3 position of phosphatidylinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3). Given the importance of PTEN in regulating PtdIns-3,4,5-P3 levels, we used Affymetrix GeneChip arrays to identify genes regulated by PTEN. PTEN expression rapidly reduced the activity of Akt, which was followed by a G(1) arrest and eventually apoptosis. The gene encoding insulin receptor substrate 2 (IRS-2), a mediator of insulin signaling, was found to be the most induced gene at all time points. A PI3K-specific inhibitor, LY294002, also upregulated IRS-2, providing evidence that it was the suppression of the PI3K pathway that was responsible for the message upregulation. In addition, PTEN, LY294002, and rapamycin, an inhibitor of mammalian target of rapamycin, caused a reduction in the molecular weight of IRS-2 and an increase in the association of IRS-2 with PI3K. Apparently, PTEN inhibits a negative regulator of IRS-2 to upregulate the IRS-2-PI3K interaction. These studies suggest that PtdIns-3,4,5-P3 levels regulate the specific activity and amount of IRS-2 available for insulin signaling.     

The PTEN-Akt pathway impacts the integrity and composition of mitotic centrosomes

Loss of the tumor suppressor PTEN is observed in many human cancers that display increased chromosome instability and aneuploidy. The subcellular fractions of PTEN are associated with different functions that regulate cell growth, invasion and chromosome stability. In this study, we show a novel role for PTEN in regulating mitotic centrosomes. PTEN localization at mitotic centrosomes peaks between prophase and metaphase, paralleling the centrosomal localization of PLK-1 and γ-tubulin and coinciding with the time frame of centrosome maturation. In primary keratinocytes, knockdown of PTEN increased whole-cell levels of γ-tubulin and PLK-1 in an Akt-dependent manner and had little effect on recruitment of either protein to mitotic centrosomes. Conversely, knockdown of PTEN reduced centrosomal levels of pericentrin in an Akt-independent manner. Inhibition of Akt activation with MK2206 reduced the whole-cell and centrosome levels of PLK-1 and γ-tubulin and also prevented the recruitment of PTEN to mitotic centrosomes. This reduction in centrosome-associated proteins upon inhibition of Akt activity may contribute to the increase in defects in centrosome number and separation observed in metaphase cells. Concomitant PTEN knockdown and Akt inhibition reduced the frequency of metaphase cells with centrosome defects when compared with MK2206 treatment alone, indicating that both PTEN and pAkt are required to properly regulate centrosome composition during mitosis. The findings presented in this study demonstrate a novel role for PTEN and Akt in controlling centrosome composition and integrity during mitosis and provide insight into how PTEN functions as a multifaceted tumor suppressor.     

Apoptosis and inactivation of the PI3-kinase pathway by tetrocarcin A in breast cancers

A survival kinase, Akt, is a downstream factor in the phosphatidylinositide-3'-kinase-dependent pathway, which mediates many biological responses including glucose uptake, protein synthesis and the regulation of proliferation and apoptosis, which is assumed to contribute to acquisition of malignant properties of human cancers. Here we find that an anti-tumor antibiotic, tetrocarcin A, directly induces apoptosis of human breast cancer cells. The apoptosis is accompanied by the activation of a proteolytic cascade of caspases including caspase-3 and -9, and concomitantly decreases phosphorylation of Akt, PDK1, and PTEN, a tumor suppressor that regulates the activity of Akt through the dephosphorylation of polyphosphoinositides. Tetrocarcin A affected neither expression of Akt, PDK1, or PTEN, nor did it affect the expression of Bcl family members including Bcl-2, Bcl-X(L), and Bax. These results suggest that tetrocarcin A could be a potent chemotherapeutic agent for human breast cancer targeting the phosphatidylinositide-3'-kinase/Akt signaling pathway.     

Hypomorphic mutation of PDK1 suppresses tumorigenesis in PTEN(+/-) mice

Many cancers possess elevated levels of PtdIns(3,4,5)P(3), the second messenger that induces activation of the protein kinases PKB/Akt and S6K and thereby stimulates cell proliferation, growth, and survival. The importance of this pathway in tumorigenesis has been highlighted by the finding that PTEN, the lipid phosphatase that breaks down PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), is frequently mutated in human cancer. Cells lacking PTEN possess elevated levels of PtdIns(3,4,5)P(3), PKB, and S6K activity and heterozygous PTEN(+/-) mice develop a variety of tumors. Knockout of PKBalpha in PTEN-deficient cells reduces aggressive growth and promotes apoptosis, whereas treatment of PTEN(+/-) mice with rapamycin, an inhibitor of the activation of S6K, reduces neoplasia. We explored the importance of PDK1, the protein kinase that activates PKB and S6K, in mediating tumorigenesis caused by the deletion of PTEN. We demonstrate that reducing the expression of PDK1 in PTEN(+/-) mice, markedly protects these animals from developing a wide range of tumors. Our findings provide genetic evidence that PDK1 is a key effector in mediating neoplasia resulting from loss of PTEN and also validate PDK1 as a promising anticancer target for the prevention of tumors that possess elevated PKB and S6K activity.     

Nuclear PTEN-mediated growth suppression is independent of Akt down-regulation

The tumor suppressor gene PTEN is a phosphoinositide phosphatase that is inactivated by deletion and/or mutation in diverse human tumors. Wild-type PTEN is expressed both in the cytoplasm and nucleus in normal cells, with a preferential nuclear localization in differentiated or resting cells. To elucidate the relationship between PTEN's subcellular localization and its biologic activities, we constructed different PTEN mutants that targeted PTEN protein into different subcellular compartments. Our data show that the subcellular localization patterns of a PTEN (deltaPDZB) mutant versus a G129R phosphatase mutant were indistinguishable from those of wild-type PTEN. In contrast, the Myr-PTEN mutant demonstrated an enhanced association with the cell membrane. We found that nuclear PTEN alone is capable of suppressing anchorage-independent growth and facilitating G1 arrest in U251MG cells without inhibiting Akt activity. Nuclear compartment-specific PTEN-induced growth suppression is dependent on possessing a functional lipid phosphatase domain. In addition, the down-regulation of p70S6K could be mediated, at least in part, through activation of AMP-activated protein kinase in an Akt-independent fashion. Introduction of a constitutively active mutant of Akt, Akt-DD, only partially rescues nuclear PTEN-mediated growth suppression. Our collective results provide the first direct evidence that PTEN can contribute to G1 growth arrest through an Akt-independent signaling pathway.     

Binding and phosphorylation of par-4 by akt is essential for cancer cell survival

Activation of the PI3K-Akt pathway by loss of tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) function, increased growth factor signaling, or oncogene expression renders cancer cells resistant to apoptotic signals and promotes tumor growth. Although Akt acts as a global survival signal, the molecular circuits of this pathway have not been completely established. We report that Akt physically binds to the pro-apoptotic protein Par-4 via the Par-4 leucine zipper domain and phosphorylates Par-4 to inhibit apoptosis. Suppression of Akt activation by the PI3K-inhibitor PTEN or LY294002, Akt expression by RNA-interference, or Akt function by dominant-negative Akt caused apoptosis in cancer cells. Apoptosis induced by inhibiting Akt was blocked by inhibition of Par-4 expression, but not by inhibition of other apoptosis agonists that are Akt substrates, suggesting that inhibition of the PI3K-Akt pathway leads to Par-4-dependent apoptosis. Thus, Par-4 is essential for PTEN-inducible apoptosis, and inactivation of Par-4 by Akt promotes cancer cell survival.     

Direct identification of PTEN phosphorylation sites

The PTEN tumor suppressor gene encodes a phosphatidylinositol 3'-phosphatase that is inactivated in a high percentage of human tumors, particularly glioblastoma, melanoma, and prostate and endometrial carcinoma. Previous studies showed that PTEN is a seryl phosphoprotein and a substrate of protein kinase CK2 (CK2). However, the sites in PTEN that are phosphorylated in vivo have not been identified directly, nor has the effect of phosphorylation on PTEN catalytic activity been reported. We used mass spectrometric methods to identify Ser(370) and Ser(385) as in vivo phosphorylation sites of PTEN. These sites also are phosphorylated by CK2 in vitro, and phosphorylation inhibits PTEN activity towards its substrate, PIP3. We also identify a novel in vivo phosphorylation site, Thr(366). Following transient over-expression, a fraction of CK2 and PTEN co-immunoprecipitate. Moreover, pharmacological inhibition of CK2 activity leads to decreased Akt activation in PTEN+/+ but not PTEN-/- fibroblasts. Our results contrast with previous assignments of PTEN phosphorylation sites based solely on mutagenesis approaches, suggest that CK2 is a physiologically relevant PTEN kinase, and raise the possibility that CK2-mediated inhibition of PTEN plays a role in oncogenesis.     

Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity

It is widely accepted that reactive oxygen species (ROS) promote tumorigenesis. However, the exact mechanisms are still unclear. As mice lacking the peroxidase peroxiredoxin1 (Prdx1) produce more cellular ROS and die prematurely of cancer, they offer an ideal model system to study ROS‐induced tumorigenesis. Prdx1 ablation increased the susceptibility to Ras‐induced breast cancer. We, therefore, investigated the role of Prdx1 in regulating oncogenic Ras effector pathways. We found Akt hyperactive in fibroblasts and mammary epithelial cells lacking Prdx1. Investigating the nature of such elevated Akt activation established a novel role for Prdx1 as a safeguard for the lipid phosphatase activity of PTEN, which is essential for its tumour suppressive function. We found binding of the peroxidase Prdx1 to PTEN essential for protecting PTEN from oxidation‐induced inactivation. Along those lines, Prdx1 tumour suppression of Ras‐ or ErbB‐2‐induced transformation was mediated mainly via PTEN.     

Expression,purification, characterization and homology modeling of active Akt/PKB,a key enzyme involved in cell survival signaling

Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its activation has been linked to tumorigenesis. Up-regulation of Akt as well as its upstream regulator phosphatidylinositol-3 kinase (PI3K) has been found in many tumors and the negative regulator of this pathway PTEN/MMAC is a tumor suppressor. As a target for drug discovery, we have expressed and purified an active Akt1 enzyme from a recombinant baculovirus-infected Sf9 cell culture. Coexpression of Akt1 with the catalytic subunit of PI3K or treatment with okadaic acid during expression was found to generate an active enzyme in the insect cell culture system. We have optimized the kinase activity and developed a simple quantitative kinase assay using biotinylated peptide substrates. Using the purified active enzyme, we have characterized its physical, catalytic and kinetic properties. Since Akt is closely related to protein kinase C (PKC) and protein kinase A, the issue of obtaining selective inhibitors of this enzyme was addressed by comparison of the structures of catalytic domains of Akt and PKC, derived by homology modeling methods. A number of amino acid differences in the ATP binding regions of these kinases were identified, suggesting that selective inhibitors of Akt can be discovered. However, the ATP binding regions are highly conserved in the three isoforms of Akt implying that the discovery of isoform-selective inhibitors would be very challenging.