Tumor Suppressor Phosphatase Research Articles

Inhibition of Phosphatidylinositol-3-OH Kinase/Akt Signaling Impairs DNA Repair in Glioblastoma Cells following Ionizing Radiation

Radiation therapy is a mainstay in the treatment of glioblastomas, but these tumors are often associated with radioresistance. Activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway, which occurs frequently in glioblastomas due to inactivation of the tumor suppressor phosphatase and tensin homologue (PTEN), correlates with radioresistance. To directly test the link between Akt activation and radioresistance, we utilized PTEN-deficient U251 glioblastoma cells engineered to inducibly restore PTEN upon exposure to doxycycline. These cells showed high basal levels of Akt activation (i.e. high levels of phospho-Akt), but induction of PTEN led to substantially decreased phospho-Akt and was associated with radiosensitization. To investigate whether the PTEN-induced radiosensitization was attributable to impaired sensing versus repair of DNA damage, we assessed levels of gamma-H2AX after ionizing radiation in U251 cells induced for PTEN. Initial post-radiation levels of gamma-H2AX foci were not decreased in PTEN-induced cells; however, the resolution of these foci was significantly delayed. In contrast to these results, induction of phosphatase-dead PTEN showed no appreciable effect. Finally, exposure of cells to the PI3K inhibitor LY294002 did not decrease the occurrence of gamma-H2AX foci after irradiation but did markedly delay their resolution. These results together support a direct link between Akt activation, repair of DNA damage, and radioresistance in glioblastoma. Targeting the PI3K/Akt pathway may modulate DNA repair to improve the efficacy of radiation therapy.

Determining Risk of Biochemical Recurrence in Prostate Cancer by Immunohistochemical Detection of PTEN Expression and Akt Activation

A considerable fraction of patients who undergo radical prostatectomy as treatment for primary prostate cancer experience biochemical recurrence detected by elevated serum levels of prostate-specific antigen. In this study, we investigate whether loss of expression of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and the phosphorylated form of the cell survival protein Akt (pAkt) predicts biochemical recurrence. Expression of PTEN and pAkt was detected by immunohistochemistry in paraffin-embedded prostate cancer tissue obtained from men undergoing radical prostatectomy. Outcome was determined by 60-month follow-up determining serum prostate-specific antigen levels. By itself, PTEN was not a good predictor of biochemical recurrence; however, in combination with pAkt, it was a better predictor of the risk of biochemical recurrence compared with pAkt alone. Ninety percent of all cases with high pAkt and negative PTEN were recurrent whereas 88.2% of those with low pAkt and positive PTEN were nonrecurrent. In addition, high Gleason scores resulted in reduced protection from decreased pAkt and increased PTEN. By univariate logistic regression, pAkt alone gives an area under the receiver-operator characteristic curve of 0.82 whereas the area under the receiver-operator characteristic curve for the combination of PTEN, pAkt, and Gleason based on a stepwise selection model is 0.89, indicating excellent discrimination. Our results indicate that loss of PTEN expression, together with increased Akt phosphorylation and Gleason score, is of significant predictive value for determining, at the time of prostatectomy, the risk of biochemical recurrence.

Phosphoinositide phosphatases in a network of signalling reactions

Phosphoinositide phosphatases dephosphorylate the three positions (D-3, 4 and 5) of the inositol ring of the poly-phosphoinositides. They belong to different families of enzymes. The PtdIns(3,4)P(2) 4-phosphatase family, the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN), SAC1 domain phosphatases and myotubularins belong to the tyrosine protein phosphatases superfamily. They share the presence of a conserved cysteine residue in the consensus CX(5)RT/S. Another family consists of the inositol polyphosphate 5-phosphatase isoenzymes. The importance of these phosphoinositide phosphatases in cell regulation is illustrated by multiple examples of their implications in human diseases such as Lowe syndrome, X-linked myotubular myopathy, cancer, diabetes or bacterial infection.

PTEN, a general negative regulator of cyclin D expression

The tumor-suppressor phosphatase with tensin homology (PTEN) is frequently mutated in many malignancies and is one of the most well studied tumor suppressor genes 1, 2. PTEN, a lipid and protein dual phosphatase, plays a vital role in embryonic development, cell growth, apoptosis and cell migration. The well-known function of PTEN is phosphatidylinositol-3 (PI3)-phosphatase, which functions as a negative regulator of the PI3 kinase (PI3K) pathway. It is well established that PTEN regulates the G1-S transition by modulating the expression of cyclin D1 and p27Kip1.

Akt activation by arachidonic acid metabolism occurs via oxidation and inactivation of PTEN tumor suppressor

Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes are overexpressed during inflammation and multistage tumor progression in many neoplastic disorders including lung, breast and pancreatic cancers. Here we report that the tumor suppressor phosphatase and tensin homolog (PTEN) is oxidized and inactivated during arachidonic acid (AA) metabolism in pancreatic cancer cell lines expressing COX-2 or 5-LOX. Oxidation of PTEN decreases its phosphatase activity, favoring increased phosphatidylinositol 3,4,5-triphosphate production, activation of Akt and phosphorylation of downstream Akt targets including GSK-3beta and S6K. These effects are recapitulated with pancreatic phospholipase A(2), which hydrolyses the release of membrane-bound AA. Interference with PTEN's physiological antagonism of signals from growth factors, insulin and oncogenes may confer risk for hypertrophic or neoplastic diseases associated with chronic inflammation or unwarranted oxidative metabolism of essential fatty acids.

Inhibition of B56-containing Protein Phosphatase 2As by the Early Response Gene IEX-1 Leads to Control of Akt Activity

The importance of PP2A in the regulation of Akt/PKB activity has long been recognized but the nature of the holoenzyme involved and the mechanisms controlling dephosphorylation are not yet known. We identified IEX-1, an early gene product with proliferative and survival activities, as a specific inhibitor of B56 regulatory subunit-containing PP2A. IEX-1 inhibits B56-PP2A activity by allowing the phosphorylation of B56 by ERK. This leads to sustained ERK activation. IEX-1 has no effect on PP2A containing other B family subunits. Thus, studying IEX-1 contribution to signaling should help the discovery of new pathways controlled by B56-PP2A. By using overexpression and RNA interference, we show here that IEX-1 increases Akt/PKB activity in response to various growth factors by preventing Akt dephosphorylation on both Thr(308) and Ser(473) residues. PP2A-B56beta and gamma subunits have the opposite effect and reverse IEX-1-mediated Akt activation. The effect of IEX-1 on Akt is ERK-dependent. Indeed: (i) a IEX-1 mutant deficient in ERK binding had no effect on Akt; (ii) ERK dominant-negative mutants reduced IEX-1-mediated increase in pAkt; (iii) a B56beta mutant that cannot be phosphorylated in the ERK.IEX-1 complex showed an enhanced ability to compete with IEX-1. These results identify B56-containing PP2A holoenzymes as Akt phosphatases. They suggest that IEX-1 behaves as a general inhibitor of B56 activity, enabling the control of both ERK and Akt signaling downstream of ERK.

Substrate specificity and acute regulation of the tumour suppressor phosphatase, PTEN

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumour suppressor that functions as a PtdIns(3,4,5)P3 3-phosphatase to inhibit cell proliferation, survival and growth by antagonizing PI3K (phosphoinositide 3-kinase)-dependent signalling. Recent work has begun to focus attention on potential biological functions of the protein phosphatase activity of PTEN and on the possibility that some of its functions are phosphatase-independent. We discuss here the structural and regulatory mechanisms that account for the remarkable specificity of PTEN with respect to its PtdIns substrates and how it avoids the soluble headgroups of PtdIns that occur commonly in cells. Secondly we discuss the concept of PTEN as a constitutively active enzyme that is subject to negative regulation both physiologically and pathologically. Thirdly, we review the evidence that PTEN functions as a dual specificity phosphatase with discrete lipid and protein substrates. Lastly we present a current model of how PTEN may participate in the control of cell migration.

PTEN-deficient intestinal stem cells initiate intestinal polyposis

Intestinal polyposis, a precancerous neoplasia, results primarily from an abnormal increase in the number of crypts, which contain intestinal stem cells (ISCs). In mice, widespread deletion of the tumor suppressor Phosphatase and tensin homolog (PTEN) generates hamartomatous intestinal polyps with epithelial and stromal involvement. Using this model, we have established the relationship between stem cells and polyp and tumor formation. PTEN helps govern the proliferation rate and number of ISCs and loss of PTEN results in an excess of ISCs. In PTEN-deficient mice, excess ISCs initiate de novo crypt formation and crypt fission, recapitulating crypt production in fetal and neonatal intestine. The PTEN-Akt pathway probably governs stem cell activation by helping control nuclear localization of the Wnt pathway effector beta-catenin. Akt phosphorylates beta-catenin at Ser552, resulting in a nuclear-localized form in ISCs. Our observations show that intestinal polyposis is initiated by PTEN-deficient ISCs that undergo excessive proliferation driven by Akt activation and nuclear localization of beta-catenin.

Substrate specificity and acute regulation of the tumour suppressor phosphatase, PTEN

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumour suppressor that functions as a PtdIns(3,4,5)P3 3-phosphatase to inhibit cell proliferation, survival and growth by antagonizing PI3K (phosphoinositide 3-kinase)-dependent signalling. Recent work has begun to focus attention on potential biological functions of the protein phosphatase activity of PTEN and on the possibility that some of its functions are phosphatase-independent. We discuss here the structural and regulatory mechanisms that account for the remarkable specificity of PTEN with respect to its PtdIns substrates and how it avoids the soluble headgroups of PtdIns that occur commonly in cells. Secondly we discuss the concept of PTEN as a constitutively active enzyme that is subject to negative regulation both physiologically and pathologically. Thirdly, we review the evidence that PTEN functions as a dual specificity phosphatase with discrete lipid and protein substrates. Lastly we present a current model of how PTEN may participate in the control of cell migration.

PTEN: Its Deregulation and Tumorigenesis

The tumor suppressor phosphatase and tensin homolog (PTEN) functions as a phosphoinositide 3-phosphatase, that antagonizes phosphatidylinositol 3-kinase action, and negatively regulates cell proliferation and survival signals. Inactivation of PTEN by loss-of-function mutations gives rise to deregulated hyperproliferation of cells, leading to oncogenic transformation. Recent studies have identified a number of upstream regulatory factors for PTEN and unveiled that the impairment in the PTEN regulatory system potentially becomes a causal factor for oncogenic transformation of cells. This article will review the PTEN inactivation mechanism which is linked to human tumorigenesis, particularly focusing on recent research progress in PTEN regulators.

Apoptin studies illuminate intersection between lipidomics and tumor suppressors

Apoptin studies illuminate intersection between lipidomics and tumor suppressors

Stimulation of PI 3-kinase signaling via inhibition of the tumor suppressor phosphatase, PTEN

Stimulation of PI 3-kinase signaling via inhibition of the tumor suppressor phosphatase, PTEN

Rituximab-Mediated Upregulation of PTEN Expression and Inhibition of Both the PI3K/AKT Pathway and Pleitrophin (PTN) Expression in B-NHL: Roles in Cell Growth Inhibition and Chemosensitization.

We have recently reported that rituximab treatment of B-NHL cell lines, like Ramos, inhibited the PI3K/AKT signaling pathway and downregulated Bcl-xL expression. The role of the AKT pathway in chemosensitization was corroborated by treating Ramos cells with the AKT inhibitor, LY294002, and resulted in sensitization of the cells to drug-induced apoptosis. We have investigated a potential underlying mechanism responsible for the rituximab-mediated inhibition of the AKT pathway. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor phosphatase and functions as a negative regulator of the PI3K pathway through its phosphatase activity. We hypothesized that rituximab may upregulate PTEN expression and thus, inhibiting the AKT pathway. Treatment of Ramos cells with rituximab (20μg/ml for 20h) resulted in significant upregulation of PTEN expression (as assessed by both Western and RT-PCR). Time kinetic analysis showed that PTEN is upregulated as early as 6–9h post rituximab treatment. In addition, since rituximab inhibits cell proliferation and cell growth, we have examined the effect of rituximab on the expression of the growth factor pleitrophin (PTN). PTN is a heparin-binding and secreted growth differentiation factor that mediates various functions such as cell motility and migration, survival, growth and differentiation. Treatment of Ramos cells with rituximab inhibited PTN expression as early as 12h post treatment. The present findings suggested that rituximab-mediated induction of PTEN expression and inhibition of both the AKT pathway and PTN epxression may be interrelated and play an important role in rituximab-mediated cell growth inhibition and chemosensitization. A recent report by Li et al., (JBC, 281:10663,2006) demonstrated that PTEN null cells exhibited upregulation of PTN expression and activation of the PI3K/AKT pathway. Further, inhibition of PTN resulted in inhibition of the AKT pathway, thus establishing a feedback mechanism. Our findings with rituximab are consistent with the Li's findings'. The mechanism by which rituximab inhibits PTN expression is not clear. Reported studies have implicated the role of AP-1 in PTN transcription and in agreement, our findings have also demonstrated that rituximab inhibits AP-1. Overall, the present studies suggest novel targets modified by rituximab namely, PTN and PTEN, which can be considered for therapeutic intervention in the treatment of both rituximab-sensitive and rituximab-resistant tumors.

Mutation-Positive and Mutation-Negative Patients with Cowden and Bannayan-Riley-Ruvalcaba Syndromes Associated with Distinct 10q Haplotypes

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) encodes a tumor-suppressor phosphatase frequently mutated in both sporadic and heritable forms of human cancer. Germline mutations are associated with a number of heritable cancer syndromes that are jointly referred to as the "PTEN hamartoma tumor syndrome" (PHTS) and include Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, and Proteus-like syndrome. Germline PTEN mutations have been identified in a significant proportion of patients with PHTS; however, there are still many individuals with classic diagnostic features for whom mutations have yet to be identified. To address this, we took a haplotype-based approach and investigated the association of specific genomic regions of the PTEN locus with PHTS. We found this locus to be characterized by three distinct haplotype blocks 33 kb, 65 kb, and 43 kb in length. Comparisons of the haplotype distributions for all three blocks differed significantly among patients with PHTS and controls (P=.0098, P<.0001, and P<.0001 for blocks 1, 2, and 3, respectively). "Rare" haplotype blocks and extended haplotypes account for two-to-threefold more PHTS chromosomes than control chromosomes. PTEN mutation-negative patients are strongly associated with a haplotype block spanning a region upstream of PTEN and the gene's first intron (P=.0027). Furthermore, allelic combinations contribute to the phenotypic complexity of this syndrome. Taken together, these data suggest that specific haplotypes and rare alleles underlie the disease etiology in these sample populations; constitute low-penetrance, modifying loci; and, specifically in the case of patients with PHTS for whom traditional mutations have yet to be identified, may harbor pathogenic variant(s) that have escaped detection by standard PTEN mutation-scanning methodologies.

PTEN in the haematopoietic system and its therapeutic indications

A unique feature of the haematopoietic system is its self-renewal ability while maintaining a stable number of pluripotent haematopoietic stem cells (HSCs). Recently, two publications by Yilmaz and colleagues and Zhang and colleagues demonstrated that the loss of the tumour suppressor phosphatase and tensin homolog (PTEN) in mice disturbed the maintenance of quiescent HSCs and promoted leukemogenesis. Mammalian target of rapamycin (mTOR) inhibition with rapamycin distinctly rescued HSC development and depleted leukemic stem cells. Thus, the regulation of HSCs and leukemic cells seems to be governed by cell-context-dependent, PTEN-mediated regulation of mTOR.

Critical Role of PICT-1, a Tumor Suppressor Candidate, in Phosphatidylinositol 3,4,5-Trisphosphate Signals and Tumorigenic Transformation

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) regulates diverse cellular functions by dephosphorylating the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). Recent study revealed that PICT-1/GLTSCR2 bound to and stabilized PTEN protein in cells, implicating its roles in PTEN-governed PIP(3) signals. In this study, we demonstrate that RNA interference-mediated knockdown of PICT-1 in HeLa cells down-regulated endogenous PTEN and resulted in the activation of PIP(3) downstream effectors, such as protein kinase B/Akt. Furthermore, the PICT-1 knockdown promoted HeLa cell proliferation; however the proliferation of PTEN-null cells was not altered by the PICT-1 knockdown, suggesting its dependency on PTEN status. In addition, apoptosis of HeLa cells induced by staurosporine or serum-depletion was alleviated by the PICT-1 knockdown in the similar PTEN-dependent manner. Most strikingly, the PICT-1 knockdown in HeLa and NIH3T3 cells promoted anchorage-independent growth, a hallmark of tumorigenic transformation. Furthermore, PICT-1 was aberrantly expressed in 18 (41%) of 44 human neuroblastoma specimens, and the PICT-1 loss was associated with reduced PTEN protein expression in spite of the existence of PTEN mRNA. Collectively, these results suggest that PICT-1 plays a role in PIP(3) signals through controlling PTEN protein stability and the impairment in the PICT-1-PTEN regulatory unit may become a causative factor in human tumor(s).

The ERK1/2 pathway modulates nuclear PTEN-mediated cell cycle arrest by cyclin D1 transcriptional regulation

PTEN, a tumor suppressor phosphatase that dephosphorylates both protein and lipid substrates, is mutated in both heritable and sporadic breast cancer. Until recently, PTEN-mediated cell cycle arrest and apoptosis were thought to occur through its well-documented cytoplasmic activities. We have shown that PTEN localizes to the nucleus coincident with the G0-G1 phases of the cell cycle and that compartmentalization may regulate cell cycle progression dependent upon the down-regulation of cyclin D1. However, the mechanism for cyclin D1-dependent growth suppression by nuclear PTEN has remained largely undefined. Utilizing MCF-7 Tet-Off breast cancer cell lines stably expressing two different nuclear localization defective PTEN mutants, as well as wild-type PTEN and empty vector control cells, we demonstrate that nuclear PTEN down-regulates cyclin D1 transcription and this event is mediated by the down-regulation of MAPK specifically by nuclear localized PTEN. These results provide further evidence that nuclear PTEN plays a role through cell cycle suppression functions in regulating carcinogenesis.

Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-γ and PTEN

Wound healing is essential for maintaining the integrity of multicellular organisms. In every species studied, disruption of an epithelial layer instantaneously generates endogenous electric fields, which have been proposed to be important in wound healing. The identity of signalling pathways that guide both cell migration to electric cues and electric-field-induced wound healing have not been elucidated at a genetic level. Here we show that electric fields, of a strength equal to those detected endogenously, direct cell migration during wound healing as a prime directional cue. Manipulation of endogenous wound electric fields affects wound healing in vivo. Electric stimulation triggers activation of Src and inositol-phospholipid signalling, which polarizes in the direction of cell migration. Notably, genetic disruption of phosphatidylinositol-3-OH kinase-gamma (PI(3)Kgamma) decreases electric-field-induced signalling and abolishes directed movements of healing epithelium in response to electric signals. Deletion of the tumour suppressor phosphatase and tensin homolog (PTEN) enhances signalling and electrotactic responses. These data identify genes essential for electrical-signal-induced wound healing and show that PI(3)Kgamma and PTEN control electrotaxis.

Tumor suppressor PTEN affects tau phosphorylation, aggregation, and binding to microtubules

Neurofibrillary tangles (NFTs), consisting of abnormally hyperphosphorylated tau, are implicated in the pathogenesis of several neurodegenerative diseases including Alzheimer's disease (AD). The molecular mechanisms underlying the regulation of tau phosphorylation are largely unknown. While the PI3K/Akt pathway has been shown to regulate multiple cellular events pertinent to AD pathogenesis, potential functions of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in AD pathogenesis have not been explored. Here, we examine the effects of PTEN on tau phosphorylation, its microtubule association and formation of aggregates, and consequentially neuronal morphology. In cultured cells, overexpression of wild-type (WT) PTEN alters tau phosphorylation at several sites, increases tau-microtubule association and decreases formation of tau aggregates. In addition, the phosphatase-null PTEN increases tau aggregation and impairs tau binding to microtubule and neurite outgrowth of neurons expressing the mutant PTEN. We also found a significant loss of PTEN in AD patient brains correlated with a dramatically increased concentration of phospho-tau at Ser-214 in NFTs. Together, our results demonstrate that PTEN regulates tau phosphorylation, binding to microtubules and formation of aggregates and neurite outgrowth. These findings suggest a link between malfunction of PTEN and tauopathy, and imply PTEN as a therapeutic target for tauopathy.

Alteration and clinical relevance of PTEN expression and its correlation with survivin expression in epithelial ovarian tumors

The tumor suppressor PTEN, phosphatase and tensin homolog on chromosome 10, plays an essential role in regulating signaling pathways involved in cell growth and apoptosis and is inactivated in a wide variety of tumors. Survivin, a member of the inhibitor of apoptosis protein family (IAP), is associated with cell proliferation, and overexpressed in common human tumors. Both PTEN and survivin proteins can regulate cell cycle and apoptosis, but their biological effects are adverse. We have previously investigated the role of survivin expression in epithelial ovarian tumors. In this study, we evaluated the alteration and clinical relevance of PTEN expression and further assessed its correlation with survivin expression in epithelial ovarian tumors. Immunohistochemical analysis was performed in 103 cases of ovarian tumors, and 26 of the 103 cases were evaluated by Western blot analysis. PTEN expression was reduced from benign to malignant ovarian tumors (p=0.0003), and an inverse correlation between PTEN and survivin was found in benign, borderline, and malignant tumors (p=0.004, p=0.015 and p=0.0005, respectively). PTEN expression was significantly associated with tumor grade (p=0.001), histological subtype (p=0.037), ascites (p=0.038), and residual disease (p=0.0006). Kaplan-Meier survival analysis showed that the loss of PTEN expression was significantly associated with poor overall survival (p=0.021), and patients with PTEN(-)/survivin(+) expression had the worst prognosis among all phenotypes of PTEN/survivin expression (p=0.039). Our results suggest that the altered PTEN expression and its inverse correlation with survivin may be involved in the development and progression of ovarian tumors, and the combined detection of PTEN and survivin proteins might be more valuable in the evaluation of malignancy and prognosis in epithelial ovarian tumors.