Potent Inhibitor Of PP2A Research Articles

Inhibitory effects of rubratoxin A, a potent inhibitor of protein phosphatase 2, on the Ca2+-dependent contraction of skinned carotid artery from guinea pig.

Rubratoxin A, a potent inhibitor of PP2A, is known to suppress smooth muscle contraction. The inhibitory role of PP2A in smooth muscle contraction is still unclear. In order to clarify the regulatory mechanisms of PP2A on vascular smooth muscle contractility, we examined the effects of rubratoxin A on the Ca2+-induced contraction of β-escin skinned carotid artery preparations from guinea pigs. Rubratoxin A at 1 µM and 10 µM significantly inhibited skinned carotid artery contraction at any Ca2+ concentration. The data fitting to the Hill equation in [Ca2+]-contraction relationship indicated that rubratoxin A decreased Fmax-Ca2+ and increased [Ca2+]50, indices of Ca2+ sensitivity for the force and myosin-actin interaction, respectively. These results suggest that PP2A inhibition causes downregulation of the myosin light chain phosphorylation and direct interference with myosin-actin interaction.

CK2 Phosphorylating I2PP2A/SET Mediates Tau Pathology and Cognitive Impairment.

Casein kinase 2 (CK2) is highly activated in Alzheimer disease (AD) and is associated with neurofibrillary tangles formation. Phosphorylated SET, a potent PP2A inhibitor, mediates tau hyperphosphorylation in AD. However, whether CK2 phosphorylates SET and regulates tau pathological phosphorylation in AD remains unclear. Here, we show that CK2 phosphorylating SET at Ser9 induced tau hyperphosphorylation in AD. We found that either Aβ treatment or tau overexpression stimulated CK2 activation leading to SET Ser9 hyperphosphorylation in neurons and animal models, while inhibition of CK2 by TBB abolished this event. Overexpression of CK2 in mouse hippocampus via virus injection induced cognitive deficit associated with SET Ser9 hyperphosphorylation. Injection of SET Ser9 phosphorylation mimetic mutant induced tau pathology and behavior impairments. Conversely co-injection of non-phosphorylated SET S9A with CK2 abolished the CK2 overexpression-induced AD pathology and cognitive deficit. Together, our data demonstrate that CK2 phosphorylates SET at Ser9 leading to SET cytoplasmic translocation and inhibition of PP2A resulting in tau pathology and cognitive impairments.

Both secreted and the cellular levels of BDNF attenuated due to tau hyperphosphorylation in primary cultures of cortical neurons

Intracellular aggregation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a major neuropathological hallmark of taupathies such as Alzheimer’s disease. Okadaic acid (OKA) is a potent inhibitor of PP2A, leading to abnormal tau phosphorylation. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that is selectively downregulated in AD. In this study, we investigated the effects of OKA induced tau hyperphosphorylation on secreted and cellular levels of BDNF in primary cortical neurons that were treated with 25nM OKA. Tau phosphorylation at threonine 231 (Thr231) sites was assessed by Western blot using antibodies against phospho-Thr231. Non-phosphorylated tau protein was detected with the Tau-1 antibody. Levels of BDNF secreted to the culture medium were determined by ELISA at the 8th and 24th hours of treatment. Cellular localization and protein expression of BDNF and tau were assessed by immunofluorescent labeling and fluorescent intensity measurements at 24h of treatment. Tau hyperphosphorylation was confirmed with increase in Thr231 and the decrease in Tau-1 signals after 8h of OKA treatment, compared with the control groups, secreted BDNF levels in the OKA-treated group were significantly lower after 24h of treatment but were not significantly different at 8h of treatment. BDNF immunoreactivity was seen in cytoplasm and neurites of the neurons in control group. BDNF immunoreactivity significantly decreased in the OKA treated group and this attenuation was significant especially at neurites. Our results suggest that the decrease in BDNF secretion and the BDNF expression might depend on the disruption of microtubule structure caused by tau hyperphosphorylation.

Abstract 615: PP2A activation as a strategy for treatment of breast cancer

Abstract Background: Cells respond to stimuli by activating kinase signaling cascades that are counterbalanced by specific phosphatases so that the cell can return to a resting state. PP2A is a critical cellular phosphatase that negatively regulates multiple kinase cascades and displays strong tumor suppressor activity (Westermarck and Hahn, Trends Mol Med, 2008, 14:152-60). Inhibition of PP2A is essential for cell transformation and tumorigenesis (Chen et al., Cancer Cell, 2004, 5:127-36) and several physiological PP2A inhibitory proteins have been described. The oncoprotein SET is a potent inhibitor of PP2A that is overexpressed in cancer. We found that SET is overexpressed in 55-60% of primary breast cancer samples and identified peptides that antagonize SET, activate PP2A, reduce phosphorylation of oncoproteins, and inhibit xenograft tumor growth. Methods: Primary breast cancer samples and matched normal adjacent tissue were evaluated for overexpression of SET using qPCR, immunofluorescence staining, and Western blotting. Cytotoxicity of PP2A activation in breast cancer cell lines was assessed using the MTT assay. PP2A activity was measured using an immunprecipitation assay and cellular correlates of dephosphorylation were measured by Western blotting. Tumors in NOD/SCID mice with human breast cancer cell lines implanted into the mammary fat pad were treated with SET antagonist or a vehicle control by subcutaneous injection. Results: Assessment of SET expression was analyzed by qPCR from breast cancer samples and normal adjacent tissues. We observed greater than 3-fold increased expression of SET in 11 of 19 patients. We also observed SET overexpression in 7 of 13 triple negative breast cancer samples in a commercially available breast cancer cDNA array. SET overexpression was confirmed in breast cancer cell lines by qPCR and Western blotting. Antagonism of SET using COG449 inhibited growth of several cell lines arising from activation of PP2A. Activation of PP2A with COG449 reduced S62 phosphorylation of c-Myc and other known PP2A targets. Finally, subcutaneous treatment with 10 mg/kg COG449 significantly inhibited growth of xenografted tumors in the mammary fat pad of NOD/SCID mice. Importantly, in vivo studies in normal mice using COG449 shows no toxicity even at doses of 100 mg/kg. Conclusions: We demonstrated SET overexpression in breast cancer samples and that SET antagonism with COG449 activated PP2A. This results in cytotoxicity in breast cancer cells in vitro and reduced phosphorylation of c-Myc S62 and other PP2A targets. PP2A activation by SET antagonism inhibited tumor growth in xenograft models at concentrations that are nontoxic in normal mice to demonstrate that PP2A activation is a novel approach to breast cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 615. doi:10.1158/1538-7445.AM2011-615

Impact of Protein Phosphatase PP2A on ATRA-Induced Activation of FoxO3a In Acute Promyelocytic Leukemia Cells

AbstractAbstract 2492Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia caused by reciprocal translocations of the long arms of chromosomes 15 and 17, which prevent cellular differentiation into mature neutrophils. The translocation of the promyelocytic leukemia (PML) gene on chromosome 15 and a retinoic acid receptor α (RARα) gene on chromosome 17 generates a PML-RARα fusion protein that inhibits PML-dependent apoptotic pathways in a dominant negative fashion. This fusion protein also blocks granulocytic differentiation by direct transcriptional inhibition of retinoic acid target genes. All-trans retinoic acid (ATRA) arrests cell growth, granulocytic differentiation, and apoptosis in APL cells via proteasome-dependent degradation of PML-RARα fusion protein and subsequent PML-nuclear body (NB) formation. Although PML is the essential component of PML-NBs and functions as a tumor suppressor, disruption of PML-NBs by the PML-RARα fusion protein inhibits endogenous PML tumor-suppressive functions in APL cells. Therefore, degradation of PML-RARα fusion protein and reorganization of PML-NBs during ATRA treatment are regarded as critical cellular responses, similar to the cell growth arrest and apoptosis of leukemia cells. Recently we demonstrated that FoxO3a (also named FKHRL1), a member of the Forkhead family of transcription factors, is a key molecule for the ATRA-induced cellular responses in APL cells (Blood 2010; 115: 3787–3795). In this study, we investigated the mechanism by which FoxO3a is activated by ATRA treatment in a human promyelocytic leukemia cell line NB4. Okadaic acid, a potent PP2A inhibitor, cancelled ATRA-induced dephosphorylation of AKT and its downstream molecule FoxO3a in NB4 cells. Knockdown of endogenous PP2A by siRNA significantly enhanced phosphorylation of both AKT and FoxO3a. These results suggested that PP2A is involved in ATRA-induced dephosphorylation of AKT and FoxO3a. Concomitantly, PP2AC, a catalytic subunit of PP2A, was dephoshorylated at tyrosine 307, and phosphatase activity of PP2A increased after ATRA treatment. Co-immunoprecipitation assay revealed that PP2A constitutively and directly binds to FoxO3a. Using artificial oligopeptides, we demonstrated that enhanced PP2A activity by ATRA directly dephosphorylates phosphothreonine 32 on FoxO3a. In addition, we found that 14-3-3 epsilon binded to phosphorylated FoxO3a in the cytoplasm in the absence of ATRA. After ATRA treatment, however, dephosphorylated FoxO3a dissociated from 14-3-3 epsilon and moved into the nucleus. Confocal microscopic analysis revealed that PP2A-FoxO3a complex partially co-localized with PML-NBs in the nucleus after ATRA treatment. Together, PML orchestrates nuclear networking with PP2A and FoxO3a for ATRA-induced granulocytic differentiation and apoptosis of APL cells. Disclosures:No relevant conflicts of interest to declare.

The SET Oncogene, a Potent PP2A Inhibitor, Is Elevated in CLL and Antagonism of SET Induces Apoptosis.

Abstract 802 Background and Significance:Even though we have treatments for CLL, it remains an incurable leukemia. We need new and better treatments for this disease. The Akt kinase is usually constitutively activated (phosphorylated) in CLL, and it acts to maintain CLL cell viability. The tumor suppressor protein phosphatase 2A is important in deactivation of Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and NFkB (through IkK). We developed apoE-mimetic peptides that potently decrease phosphorylation of Akt and MAPKs, decrease TNF and nitric oxide synthase expression, and display anti-inflammatory activity in vitro and in vivo by antagonism of SET, a potent physiological inhibitor of PP2A. Others have reported that PP2A activity is reduced and that SET is overexpressed in cells of chronic myelocytic leukemia patients. Increased SET expression with consequent decreased PP2A activity leads to dysregulated kinase signaling. We show here that SET is also overexpressed in CLL cells, and that SET antagonist apoE-mimetic peptides kill CLL cells. Methods:Patients were from the Duke University and V.A. Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CD19+ CLL or normal B cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. ApoE-mimetic peptides were prepared by chemical synthesis. Western blotting was used with anti-SET and anti-beta-actin antibodies. Apoptosis assays were performed with the annexin-V:propidium iodide staining method. Results:Samples from 17 CLL patients and 5 normal volunteers were examined by Western blotting. SET protein levels were 6.2-fold higher in CLL cells than in normal B cells. The apoE-mimetic COG compounds are peptides of 17 to 34 amino acids derived from the ligand-binding region of the apolipoprotein E holoprotein, and they act by binding to SET and preventing the inhibition of PP2A. This results in a net increase in PP2A activity in CLL cells. We have examined 11 COG peptides. Each of the 11 peptides displayed some cytotoxicity for CLL cells in vitro, irrespective of the patients' stages and other good or bad prognostic findings. 12 of 17 CLL patients were Rai stage 0 at presentation, and 5 were stage 1 or 2. They had been followed 4.2 yr (median; range 1.0 – 24.1 yr). 2 of 16 were CD38 positive, and 6 of 15 were Zap-70 positive. Of 15 analyzed, 6 had unmutated IgVH gene. 11 of 17 patients had not been treated. Peptide COG449 was the most potent, while a control peptide with an inverted apoE sequence had no activity. COG449 induced cell death in a dose-dependent fashion in all patients' samples, with a mean ED50 of 80 nM. The ED50 of COG449 for normal B cells was very high (> 10,000 nM). Annexin-V staining indicated that apoptosis was induced at concentrations in good agreement with the ED50 for cytotoxicity of the compounds tested. In vivo studies in normal mice using COG449 show no toxicity even at doses of 100 mg/kg when delivered by subcutaneous injection. Conclusions:We demonstrated SET overexpression in CLL cells and that apoE-mimetic peptides bind SET to de-inhibit PP2A. This results in apoptosis and death of CLL cells in vitro with high efficacy and potency (low nanomolar ED50s). CLL cells are killed preferentially compared to normal PBMC and B cells. Preliminary studies show that the peptide is nontoxic in normal mice. Trials in CLL patients will help determine the efficacy in vivo. Disclosures:Christensen:Cognosci Inc.: Employment. Oddo:Cognosci Inc.: Employment. Vitek:Cognosci Inc.: Employment, Equity Ownership.

Synthesis and biological evaluation of norcantharidin analogues: Towards PP1 selectivity

Simple modifications to the anhydride moiety of norcantharidin have lead to the development of a series of analogues displaying modest PP1 inhibition (low μM IC 50s) comparable to that of norcantharidin (PP1 IC 50 = 10.3 ± 1.37 μM). However, unlike norcantharidin, which is a potent inhibitor of PP2A (IC 50 = 2.69 ± 1.37 μM), these analogues show reduced PP2A inhibitory action resulting in the development of selective PP1 inhibitory compounds. Data indicates that the introduction of two ortho-disposed substituents on an aromatic ring, or para-substituent favours PP1 inhibition over PP2A inhibition. Introduction of a p-morphilinoaniline substituent, 35, affords an inhibitor displaying PP1 IC 50 = 6.5 ± 2.3 μM; and PP2A IC 50 = 7.9 ± 0.82 μM (PP1/PP2A = 0.82); and a 2,4,6-trimethylaniline, 23, displaying PP1 IC 50 = 48 ± 9; and PP2A IC 5 85 ± 3 μM (PP1/PP2A = 0.56). The latter shows a 7-fold improvement in PP1 versus PP2A selectivity when compared with norcantharidin. Subsequent analysis of 23 and 35 as potential PP2B inhibitors revealed modest inhibition with IC 50s of 89 ± 6 and 42 ± 3 μM, respectively, and returned with PP1/PP2B selectivities of 0.54 and 0.15. Thus, these analogues are the simplest and most selective PP1 inhibitors retaining potency reported to date.

Reduced Protein Phosphatase 2A Activity Induces Hyperphosphorylation and Altered Compartmentalization of Tau in Transgenic Mice

Hyperphosphorylated isoforms of the microtubule-associated protein tau are the major components of neurofibrillary lesions in Alzheimer's disease (AD). Protein phosphatase (PP) 2A is a major phosphatase implicated in tau dephosphorylation in vitro. Dephosphorylation of tau can be blocked in vivo by okadaic acid, a potent inhibitor of PP2A. Moreover, activity of PP2A is reduced in AD brains. To elucidate the role of PP2A in tau phosphorylation and pathogenesis, we expressed a dominant negative mutant form of the catalytic subunit Calpha of PP2A, L199P, in mice by using a neuron-specific promoter. We obtained mice with high expression levels of Calpha L199P in cortical, hippocampal, and cerebellar neurons. PP2A activity in brain homogenates of transgenic mice was reduced to 66%. Endogenous tau protein was hyperphosphorylated at distinct sites including the AT8 epitope Ser-202/Thr-205, a major AD-associated tau phosphoepitope. AT8-positive tau aggregates accumulated in the soma and dendrites of cortical pyramidal cells and cerebellar Purkinje cells and co-localized with ubiquitin. Our data establish that PP2A plays a crucial role in tau phosphorylation. Our results also show that reduced PP2A activity is associated with altered compartmentalization and ubiquitination of tau, resembling a key pathological finding in AD.

Fostriecin, an inhibitor of protein phosphatase 2A, limits myocardial infarct size even when administered after onset of ischemia.

The role of protein phosphatases (PPs) during ischemic preconditioning in the rabbit heart was examined. Fostriecin, a potent inhibitor of PP2A, was administered to isolated rabbit hearts starting either 15 minutes before or 10 minutes after the onset of a 30-minute period of regional ischemia and continuing until the onset of reperfusion. After 2 hours of reperfusion, infarct size was measured with triphenyltetrazolium chloride. In a second study with isolated rabbit cardiomyocytes, the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of isolated control and ischemically preconditioned cells were also measured. In a third series of experiments, left ventricular biopsies of isolated rabbit hearts were obtained before and at selected times during 60 minutes of global ischemia, and the tissue was assayed for PP1 and PP2A activities. In isolated hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts (33%; P<0.001) but comparable to that in ischemically preconditioned hearts (9%; P<0.001 versus control). Significant protection was also observed in the hearts treated only after the onset of ischemia (18% infarction; P<0.05 versus control). In isolated myocytes, fostriecin also provided protection comparable to that produced by metabolic preconditioning. Preconditioning had no apparent effect on the activity of either PP1 or PP2A in isolated ventricular myocytes or ventricular tissue obtained from heart biopsies. Fostriecin, a potent inhibitor of PP2A, can protect the rabbit heart from infarction even when administered after the onset of ischemia. But inhibition of either PP1 or PP2A does not appear to be the mechanism of protection from ischemic preconditioning.

Protein Phosphatase Inhibitors Potentiate Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Rat Pancreatic Acinar Cells

Cholecystokinin (CCK) is known to rapidly and transiently increase both [Ca2+]iand autonomous CaM kinase II activity in rat pancreatic acini. Because induction of autonomous activity may involve intramolecular autophosphorylation, the effects of protein phosphatase inhibitors were examined. None of the inhibitors tested (okadaic acid, calyculin A, and cyclosporin A) affected basal activity. Okadaic acid, a potent inhibitor of PP2A and weaker inhibitor of PP1, increased the peak autonomous activity by 30% over the level normally induced by CCK alone, while calyculin A, a potent inhibitor of both PP1 and PP2A, showed an even greater increase of 97%. Both inhibitors also delayed the decline of autonomous activity and calyculin A had a more potent effect than okadaic acid. Cyclosporin A, an inhibitor of PP2B, had no effect. The data indicate that PP1 may be involved in the dephosphorylation of CaMK II and decline of autonomous activity.