Phosphatase Homolog Research Articles

Abstract 5253: PTEN represses oncogene expression by regulating Daxx-H3.3 deposition in the chromatin

Abstract One of the most mutated and deleted gene in cancer is the tumor suppressor gene pten (phosphatase and tensin homology, deleted on chromosome ten). To further understand how PTEN inactivation contributes to cancer predisposition, we investigated how PTEN regulates gene expression. We found that the histone chaperon protein Daxx (death domain-associated protein) associates with PTEN, in vivo and in vitro, and regulates PTEN stability through acetylation and ubiquitination. We observed that PTEN regulates Histone 3.3 (H3.3 variant) loading in the chromatin by limiting Daxx interactions, and thereby controls expression of several tumor driver genes. Furthermore, when Daxx expression is disrupted in pten-deficient glioma stem cells, we observed a dramatic diminution of pluripotency transcription factors, a decrease in differentiation and sphere-forming capacity, and an increase in radiation sensitivity. In conclusion, this work supports a novel chromatin-associated function for PTEN that regulates H3.3 deposition, and gene expression, through Daxx interaction. This nominates Daxx as a therapeutic target to treat patients with gliomas and other types of cancers in which PTEN is mutated. Citation Format: Jorge A. Benitez, Webster K. Cavenee, Frank F. Furnari. PTEN represses oncogene expression by regulating Daxx-H3.3 deposition in the chromatin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5253. doi:10.1158/1538-7445.AM2014-5253

Abstract 1927: Tumor suppressor PTEN regulates cancer stem cells of glioblastoma multiforme: identification of signaling pathways as targets of therapy

Abstract Glioblastoma Multiforme (GBM), a WHO defined grade IV astrocytoma, is a uniformly fatal CNS malignancy. The high rate of recurrence is attributed to the presence of its untreatable cancer stem cells (CSC). Phosphatase and tensin homologue on chromosome 10 (PTEN), a tumor suppressor is a central negative regulator of PI3K/AKT signaling axis that controls multiple cellular functions, including cell growth, survival and proliferation. Recent evidence suggests that PTEN loss is associated with increased CSC in GBM. Furthermore, downstream from PTEN/PI3K/AKT is mTOR, an atypical serine threonine kinase that exists in two multiprotein complexes (mTORC1 and mTORC2), which regulates growth and dissemination of GBM. The molecular mechanism involved in regulation of CSC by PTEN and its interacting pathways remains elusive. The aim of this study is to elucidate the role of PTEN in regulation and maintenance of CSC in GBM, and define the potential downstream targets of therapy. In order to achieve our goals, we evaluated the presence of activated AKT/mTOR, and stem cell marker nestin in surgically resected GBM tumors. GBM cell line U87 (PTEN mutated) was transfected with PTEN plasmids (wild type and 3 phosphorylated mutants) to evaluate its role in CSC self-renewal. Pharmacological treatments included an inhibitor of EGFR, gefitinib (Iressa), with or without inhibitors of PI3K (LY294002; LY) or inhibitors of mTORC1 (rapamycin) or mTORC1/2 (PP242). Expression of mTOR and stem cell markers were assessed using IHC or IF analysis, and protein expression was determined via Western Blotting. Functional analysis included self-renewal, drug withdrawal, differentiation, proliferation and migration. Results demonstrated that a significant number of GBM tumors expressed stem cell markers nestin as well as pAKT and pmTOR. Wild type PTEN expressing CSC had decreased self-renewal compared to PTEN expressing mutant CSCs. PI3K inhibitor, LY, with gefitinib was most effective in suppressing neurosphere formation. Rebound following withdrawal of inhibitors showed a significant upregulation in self-renewal of CSC in cells treated with gefitinib and either LY or PP242. These combinations also significantly suppressed CSC proliferation. mTOR suppression altered the expression of stem cell markers nestin and SOX2. In conclusion, the findings of this study demonstrate that PTEN/PI3K/mTOR signaling forms a complex network that regulates and maintains GBM CSC; this also underscores the combined suppression of interacting signaling pathways, such as EGFR, in targeting GBM CSC. Note: This abstract was not presented at the meeting. Citation Format: Michael LaBagnara, Keith Lambert, Sudeepta Sridhara, Michael Tobias, Raj Murali, Meena Jhanwar-Uniyal. Tumor suppressor PTEN regulates cancer stem cells of glioblastoma multiforme: identification of signaling pathways as targets of therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1927. doi:10.1158/1538-7445.AM2014-1927

Constitutive Photomorphogensis Protein1 (COP1) mediated p53 pathway and its oncogenic role

We have reviewed the COP1 mediated tumor suppressor protein p53 pathway and its oncogenic role. COP1 is a negative regulator of p53 and acts as a pivotal controller of p53-Akt death-live switch (Protein kinase B). In presence of p53, COP1 is overexpressed in breast, ovarian, gastric cancers, even without MDM2 (Mouse double minute-2) amplification. Following DNA damage, COP1 is phosphorylated instantly by ATM (Ataxia telangiectasia mutated) and degraded by 14-3-3σ following nuclear export and enhancing ubiquitination. In ATM lacking cell, other kinases, i.e. ATR (ataxia telangiectasia and Rad3-related protein), Jun kinases and DNA-PK (DNA-dependent protein kinase) cause COP1 & CSN3 (COP9 signalosome complex subunit-3) phosphorylation and initiate COP1’s down regulation. Although, it has been previously found that co-knockout of MDM2 and COP1 enhance p53’s half life by eight fold, the reason is still unknown. Additionally, while interacting with p53, COP1 upregulate MDM2’s E3 ubiquitin ligase, Akt, CSN6 (COP9 signalosome 6) activity and inhibit 14-3-3σ’s negative regulation on MDM2 and COP1 itself. Conclusively, there persists an amplification loop among COP1, MDM2, Akt and 14-3-3σ to regulate p53’s stability and activity. However, the role of another tumor suppressor PTEN (phosphatase and tensin homologue) is yet to be discovered. This study provides insight on the molecular genetic pathways related to cancer and might be helpful for therapeutic inventions.

O6.10 * GLIOMA CELL VEGFR-2 EXPRESSION IMPAIRS CHEMOTHERAPEUTIC AND ANTIANGIOGENIC TREATMENTS IN PTEN-DEFICIENT GLIOBLASTOMA

BACKGROUND: Given the recent failure of bevacizumab (BEV) in extending overall survival of patients with newly diagnosed glioblastoma, tissue biomarkers predicting response to antiangiogenic treatments are urgently needed. Here, we hypothesized that loss of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a prerequisite for glioma cell expression of vascular endothelial growth factor receptor (VEGFR)-2 in glioblastoma defining a more aggressive glioblastoma subgroup prone to develop evasive resistance towards antiangiogenic treatments including BEV. METHODS AND RESULTS: Besides its ubiquitous expression on endothelial cells, immunohistochemical analysis of 59 glioblastoma tissues showed glioma cell-bound VEGFR-2 expression was restricted to PTEN-deficient tumor specimens and detectable in 20% of all specimens examined, most pronounced in infiltration zones. In glioma cells, pharmacological and RNAi-mediated inhibition of VEGFR-2 signaling revealed proproliferative, antiinvasive and chemoprotective functions for VEGFR-2 in several experimental paradigms comprising flow cytometry-based cell cycle analyses, in vitro proliferation and invasion assays, organotypic brain slice culture assays, and an MRI-monitored orthotopic mouse model. VEGFR-2-dependent cellular effects were concomitant with activation of three potent resistance factors: 'kappa-light-chain-enhancer' of activated B-cells (NF-κB), protein kinase B (AKT/PKB), and N-myc downstream regulated gene (NDRG)1. Two-photon in vivo microscopy demonstrated that glioma cell expression of VEGFR-2 hampers antiangiogenesis and favors a proinvasive response to treatment with BEV. CONCLUSIONS: Glioma cell expression of VEGFR-2, next to its expression on vessels, indicates a particularly aggressive glioblastoma subgroup prone to develop early resistance to chemotherapy with temozolomide or antiangiogenic treatment with BEV. Loss of PTEN can serve as a readily available surrogate marker to identify those tumors upfront by routine neuropathological methods.

The Hexosamine Biosynthetic Pathway: A role in nutrient regulation of growth signalling in the human placenta

The Hexosamine Biosynthetic Pathway: A role in nutrient regulation of growth signalling in the human placenta

MiRNA-1297 induces cell proliferation by targeting phosphatase and tensin homolog in testicular germ cell tumor cells.

To investigate the role of miR-1297 and the tumor suppressor gene PTEN in cell proliferation of testicular germ cell tumors (TGCT). MTT assays were used to test the effect of miR-1297 on proliferation of the NCCIT testicular germ cell tumor cell line. In NCCIT cells, the expression of PTEN was assessed by Western blotting further. In order to confirm target association between miR-1297 and 3'-UTR of PTEN, a luciferase reporter activity assay was employed. Moreover, roles of PTEN in proliferation of NCCIT cells were evaluated by transfection of PTEN siRNA. Proliferation of NCCIT cells was promoted by miR-1297 in a concentration-dependent manner. In addition, miR-1297 could bind to the 3'-UTR of PTEN based on luciferase reporter activity assay, and reduced expression of PTEN at protein level was found. Proliferation of NCCIT cells was significantly enhanced after knockdown of PTEN by siRNA. miR-1297 as a potential oncogene could induce cell proliferation by targeting PTEN in NCCIT cells.

Arabidopsis CPL4 is an essential C-terminal domain phosphatase that suppresses xenobiotic stress responses.

Eukaryotic gene expression is both promoted and inhibited by the reversible phosphorylation of the C-terminal domain of RNA polymerase II (pol II CTD). More than 20 Arabidopsis genes encode CTD phosphatase homologs, including four CTD phosphatase-like (CPL) family members. Although in vitro CTD phosphatase activity has been established for some CPLs, none have been shown to be involved in the phosphoregulation of pol II in vivo. Here we report that CPL4 is a CTD phosphatase essential for the viability of Arabidopsis thaliana. Mass spectrometry analysis identified the pol II subunits RPB1, RPB2 and RPB3 in the affinity-purified CPL4 complex. CPL4 dephosphorylates both Ser2- and Ser5-PO(4) of the CTD in vitro, with a preference for Ser2-PO(4). Arabidopsis plants overexpressing CPL4 accumulated hypophosphorylated pol II, whereas RNA interference-mediated silencing of CPL4 promoted hyperphosphorylation of pol II. A D128A mutation in the conserved DXDXT motif of the CPL4 catalytic domain resulted in a dominant negative form of CPL4, the overexpression of which inhibited transgene expression in transient assays. Inhibition was abolished by truncation of the phosphoprotein-binding Breast Cancer 1 C-terminal domain of CPL4, suggesting that both catalytic function and protein-protein interaction are essential for CPL4-mediated regulation of gene expression. We were unable to recover a homozygous cpl4 mutant, probably due to the zygotic lethality of this mutation. The reduction in CPL4 levels in CPL4(RNAi) plants increased transcript levels of a suite of herbicide/xenobiotic-responsive genes and improved herbicide tolerance, thus suggesting an additional role for CPL4 as a negative regulator of the xenobiotic detoxification pathway.

A role for eisosomes in maintenance of plasma membrane phosphoinositide levels.

The plasma membrane delineates the cell and mediates its communication and material exchange with the environment. Many processes of the plasma membrane occur through interactions of proteins with phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P2), which is highly enriched in this membrane and is a key determinant of its identity. Eisosomes function in lateral organization of the plasma membrane, but the molecular function of their major protein subunits, the BAR domain-containing proteins Pil1 and Lsp1, is poorly understood. Here we show that eisosomes interact with the PI(4,5)P2 phosphatase Inp51/Sjl1, thereby recruiting it to the plasma membrane. Pil1 is essential for plasma membrane localization and function of Inp51 but not for the homologous phosphatidylinositol bisphosphate phosphatases Inp52/Sjl2 and Inp53/Sjl3. Consistent with this, absence of Pil1 increases total and available PI(4,5)P2 levels at the plasma membrane. On the basis of these findings, we propose a model in which the eisosomes function in maintaining PI(4,5)P2 levels by Inp51/Sjl1 recruitment.

Energy, the driving force behind good and ill health.

Energy, the driving force behind good and ill health.

Formation of protein S-nitrosylation by reactive oxygen species

In the present study, the formation of whole cellular S-nitrosylated proteins (protein-SNOs) by the reactive oxygen species (ROS), hydrogen peroxide (H2O2), and superoxide (O2•−) is demonstrated. A spectrum of protein cysteine oxidative modifications was detected upon incubation of serum-starved mouse embryonic fibroblasts with increasing concentrations of exogenous H2O2, ranging from exclusive protein-SNOs at low concentrations to a mixture of protein-SNOs and other protein oxidation at higher concentrations to exclusively non-SNO protein oxidation at the highest concentrations of the oxidant used. Furthermore, formation of protein-SNOs was also detected upon inhibition of the antioxidant protein Cu/Zn superoxide dismutase that results in an increase in intracellular concentration of O2•−. These results were further validated using the phosphatase and tensin homologue, PTEN, as a model of a protein sensitive to oxidative modifications. The formation of protein-SNOs by H2O2 and O2•− was prevented by the NO scavenger, c-PTIO, as well as the peroxinitrite decomposition catalyst, FETPPS, and correlated with the production or the consumption of nitric oxide (NO), respectively. These data suggest that the formation of protein-SNOs by H2O2 or O2•− requires the presence or the production of NO and involves the formation of the nitrosylating intermediate, peroxinitrite.

Switching on ubiquitylation by phosphorylating a ubiquitous activator

The dysfunction of the E3 ubiquitin ligase Parkin is a key contributor to the development of early-onset Parkinson's disease. Parkin is responsible for the labelling of outer mitochondrial membrane proteins with the small modifier protein ubiquitin in response to oxidative stress. This ubiquitylation signals the clearance of the damaged mitochondria to preserve overall cell health. Recent structural and biochemical experiments have shown that native Parkin exists in an autoinhibited state that must be activated in order to unmask its full ubiquitylation potential. In a recent article in the Biochemical Journal (vol. 460, pp. 127-139), Kazlauskaite and co-workers identified that the Parkinson's disease-associated kinase PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1] can phosphorylate ubiquitin in response to mitochondrial depolarization. Furthermore, the authors demonstrated that phosphorylated ubiquitin can activate Parkin's E3 ligase activity and promote both increased autoubiquitylation and substrate ubiquitylation of the mitochondrial protein Miro1. The study provides exciting initial insights that show how PINK1 might activate ubiquitin through phosphorylation, and how this important regulatory step might switch on Parkin-mediated ubiquitylation.

Loss of PP2A and PTEN immunoexpression coexists with survivin overexpression in adenomyosis

Phosphatase and tensin homolog (PTEN) and protein phosphatase type 2A (PP2A) are negative modulators of PI3K/AKT/survivin signaling. To evaluate immunoexpression of PTEN, PP2A and survivin in adenomyosis, ectopic lesions from 28 patients with adenomyosis and endometria from 30 controls without adenomyosis were employed in the study. The expression of PTEN, PP2A and survivin was examined with the use of immunohistochemistry. We found a decreased expression of PP2A and PTEN in adenomyosis. The expression of PTEN showed great individual differences in adenomyosis, although expression of both PP2A and PTEN was lower in adenomyosis than in normal endometria. In contrast, the expression of survivin was higher in adenomyosis. Our results suggest the important role of the PI3K cascade in the pathogenesis and development of adenomyosis.

Expression and significance of PTEN and HIF-1α proteins in sinonasal inverted papilloma

To investigate the expression and clinical significance of phosphatase and tensin homology deleted on chromosome ten (PTEN) and hypoxia-inducible factor 1 alpha (HIF-1α) in sinonasal inverted papilloma (SNIP) of different pathological grades. Fifty-five paraffin samples from patients with SNIP and a control group of 10 paraffin samples of patients with normal nasal cavity mucosa (NM) who underwent inferior turbinectomy were consisted in this study. Among the 55 cases of SNIP, 30 cases were without dysplasia subtypes, 11 cases were with dysplasia subtypes, and 14 cases with canceration to squamous cell carcinoma (SCC) subtypes. PTEN and HIF-1α expression in SNIP was detected by immunohistochemistry. The differences between NM and SNIP, and among the three subtypes were analyzed, and the relationship between PTEN, HIF-1α expression and SNIP recurrence and the correlation between PTEN expression and HIF-1α expression were also analyzed. SPSS 16.0 software was used to analyze the data. The positive expression rate of PTEN in NM and SNIP was 100% and 65.5%, the difference was significant (U = 147, P = 0.014), while HIF-1α was 0 and 30.9%, the difference was significant (U = 190, P = 0.045). The positive expression rate of PTEN in SNIP without dysplasia, SNIP with dysplasia and NSCC was 83.3%, 63.6%, 28.6%, respectively, the difference was significant (H = 12.644, P = 0.002); while HIF-1α was 16.7%, 45.5%, 50.0%, respectively, the difference was significant (H = 8.292, P = 0.016). A total of 22 SNIP patients recurred. PTEN had lower expression in recurrent SNIP (45.5%) than that in non-recurrent SNIP (82.8%), and the difference was significant(χ² = 7.834, P = 0.005). However, the expression of HIF-1α had no significant difference between recurrent SNIP and the SNIP which had no recurrence (χ² = 0.901, P = 0.343). The expression of PTEN protein was negatively correlated with that of HIF-1α protein (r = -0.503, P = 0.001). PTEN expression decreased graduately with the severity of malignancy of SNIP, but HIF-1α increased. The expression of HIF-1α was induced by hypoxia, which may negatively effect the expression of PTEN, and both HIF-1α and PTEN may play critical roles in the progress of SNIP. PTEN is one of the factors responsible for the postoperative recurrence of SNIP.

595 Deletion of Phosphatase and Tensin Homolog PTEN in the Intestinal Epithelium Causes Spontaneous Early-Onset Colitis in an IL-10 Deficient Environment

595 Deletion of Phosphatase and Tensin Homolog PTEN in the Intestinal Epithelium Causes Spontaneous Early-Onset Colitis in an IL-10 Deficient Environment

The TRAF3 adaptor protein drives proliferation of anaplastic large cell lymphoma cells by regulating multiple signaling pathways

T cells devoid of tumor necrosis factor receptor associated factor-3 (Traf3) exhibit decreased proliferation, sensitivity to apoptosis, and an improper response to antigen challenge. We therefore hypothesized that TRAF3 is critical to the growth of malignant T cells. By suppressing TRAF3 protein in different cancerous T cells, we found that anaplastic large cell lymphoma (ALCL) cells require TRAF3 for proliferation. Since reducing TRAF3 results in aberrant activation of the noncanonical nuclear factor-κB (NF-κB) pathway, we prevented noncanonical NF-κB signaling by suppressing RelB together with TRAF3. This revealed that TRAF3 regulates proliferation independent of the noncanonical NF-κB pathway. However, suppression of NF-κB-inducing kinase (NIK) along with TRAF3 showed that high levels of NIK have a partial role in blocking cell cycle progression. Further investigation into the mechanism by which TRAF3 regulates cell division demonstrated that TRAF3 is essential for continued PI3K/AKT and JAK/STAT signaling. In addition, we found that while NIK is dispensable for controlling JAK/STAT activity, NIK is critical to regulating the PI3K/AKT pathway. Analysis of the phosphatase and tensin homolog (PTEN) showed that NIK modulates PI3K/AKT signaling by altering the localization of PTEN. Together our findings implicate TRAF3 as a positive regulator of the PI3K/AKT and JAK/STAT pathways and reveal a novel function for NIK in controlling PI3K/AKT activity. These results provide further insight into the role of TRAF3 and NIK in T cell malignancies and indicate that TRAF3 differentially governs the growth of B and T cell cancers.

Modulation of the sensitivity of Jurkat T-cells to inhibition of protein synthesis by tumor necrosis factor α-related apoptosis-inducing ligand.

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in Jurkat T lymphoma cells. One of the characteristics of the phase preceding overt apoptosis is the marked downregulation of protein synthesis. We have investigated factors that can influence this response and have explored some of the signaling pathways involved. We show that interferon-α (IFNα) pretreatment desensitizes Jurkat cells to TRAIL-induced inhibition of protein synthesis, such that the concentration of TRAIL required for 50% inhibition is increased by 10-fold. The inhibition of translation is characterized by dephosphorylation of the eIF4E-binding protein 4E-BP1 and IFNα desensitizes Jurkat cells to this effect. IFNα also inhibits TRAIL-mediated dephosphorylation of the growth-promoting protein kinase B (Akt). Since Jurkat cells are defective for phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and therefore have constitutive phosphoinositide 3-kinase (PI3K) activity, we investigated the consequences for protein synthesis of inhibiting PI3K using LY294002. Inhibition of PI3K partially inhibits translation, but also enhances the effect of a suboptimal concentration of TRAIL. However, LY294002 does not block the ability of IFNα to protect protein synthesis from TRAIL-induced inhibition. Data are presented suggesting that IFNα impairs the process of activation of caspase-8 within the TRAIL death-inducing signaling complex.

MP34-09 C-TERMINAL HYPER-PHOSPHORYLATION OF PTEN IS A MAJOR MECHANISM OF PTEN INACTIVATION IN BLADDER CANCER: IMPLICATIONS IN TUMOR SUPPRESSION AND MOLECULAR THERAPY

INTRODUCTION AND OBJECTIVES: Phosphatase and Tensin Homology (PTEN) antagonizes PI3-kinase/AKT and this activity relies on PTEN-membrane interaction. While loss of PTEN, along with p53 defects, triggers muscle-invasive bladder cancer (BC) in genetically engineered mice, PTEN deletion or mutation in human BC is rare, suggesting that PTEN is inactivated via a different mechanism. Hyperphosphorylation of PTEN’s C-terminus (PTEN-Cp) is thought to dissociate PTEN from the membrane, thus functionally inactivating PTEN. This study investigates the prevalence of PTEN-Cp and its effects on tumor suppression in BC cells. METHODS: Ten cell lines representing different grades/stages of human BC was assessed for PTEN-Cp using site-specific antibodies. cDNAs encoding (i) wild-type PTEN, (ii) mutated PTEN devoid of C-terminal phosphorylation sites and (iii) truncated PTEN lacking the C-terminus were transfected into UMUC3 and J82 BC cell lines that lack the PTEN gene. PTEN-Cp status, PI3K/AKT activation and BC cell proliferation were assessed in vitro and in vivo using xenograft models. PTEN-Cp was also assessed using Western blotting of freshly preserved human BC tissues. RESULTS: Prominent PTEN-Cp was observed in all BC cell lines except UMUC3 and J82 which lack the PTEN gene. Transfection of wild-type PTEN into UMUC3 and J82 inhibited cell growth, albeit only at serum-free or low-serum (2%) conditions. High-serum (10%) caused PTEN to undergo extensive PTEN-Cp, increased AKT activation and cell proliferation. Mutated PTEN devoid of C-terminal phosphorylation sites and truncated PTEN lacking the C-terminus exerted much greater inhibitory effects on AKT and cell growth of UMUC3 and J82. These effects were reproducible when the transfected cells were transplanted subcutaneously into xenograft models. Finally, surveying a cohort of fresh human BC tissues revealed that a great majority of lowgrade non-invasive and high-grade invasive BC expressed PTEN and whenever PTEN was expressed it was highly phosphorylated at the C-terminus. CONCLUSIONS: Our studies provide the first evidence demonstrating that hyper-phosphorylation at the C-terminus is a major mechanism of PTEN inactivation in human BC, that PTEN phosphorylation may serve as a useful biomarker for BC progression, and that the tail-less PTEN is a much more potent tumor suppressor than the fulllength PTEN when used in molecular therapy.

MP34-10 CROSSTALK BETWEEN DOWNSTREAM EFFECTORS OF RECEPTOR TYROSINE KINASE/RAS PATHWAY CAN BE DISRUPTED BY COMBINATORIAL INHIBITION: IMPLICATION IN TARGETED THERAPY

INTRODUCTION AND OBJECTIVES: Phosphatase and Tensin Homology (PTEN) antagonizes PI3-kinase/AKT and this activity relies on PTEN-membrane interaction. While loss of PTEN, along with p53 defects, triggers muscle-invasive bladder cancer (BC) in genetically engineered mice, PTEN deletion or mutation in human BC is rare, suggesting that PTEN is inactivated via a different mechanism. Hyperphosphorylation of PTEN’s C-terminus (PTEN-Cp) is thought to dissociate PTEN from the membrane, thus functionally inactivating PTEN. This study investigates the prevalence of PTEN-Cp and its effects on tumor suppression in BC cells. METHODS: Ten cell lines representing different grades/stages of human BC was assessed for PTEN-Cp using site-specific antibodies. cDNAs encoding (i) wild-type PTEN, (ii) mutated PTEN devoid of C-terminal phosphorylation sites and (iii) truncated PTEN lacking the C-terminus were transfected into UMUC3 and J82 BC cell lines that lack the PTEN gene. PTEN-Cp status, PI3K/AKT activation and BC cell proliferation were assessed in vitro and in vivo using xenograft models. PTEN-Cp was also assessed using Western blotting of freshly preserved human BC tissues. RESULTS: Prominent PTEN-Cp was observed in all BC cell lines except UMUC3 and J82 which lack the PTEN gene. Transfection of wild-type PTEN into UMUC3 and J82 inhibited cell growth, albeit only at serum-free or low-serum (2%) conditions. High-serum (10%) caused PTEN to undergo extensive PTEN-Cp, increased AKT activation and cell proliferation. Mutated PTEN devoid of C-terminal phosphorylation sites and truncated PTEN lacking the C-terminus exerted much greater inhibitory effects on AKT and cell growth of UMUC3 and J82. These effects were reproducible when the transfected cells were transplanted subcutaneously into xenograft models. Finally, surveying a cohort of fresh human BC tissues revealed that a great majority of lowgrade non-invasive and high-grade invasive BC expressed PTEN and whenever PTEN was expressed it was highly phosphorylated at the C-terminus. CONCLUSIONS: Our studies provide the first evidence demonstrating that hyper-phosphorylation at the C-terminus is a major mechanism of PTEN inactivation in human BC, that PTEN phosphorylation may serve as a useful biomarker for BC progression, and that the tail-less PTEN is a much more potent tumor suppressor than the fulllength PTEN when used in molecular therapy.

Is PTEN hyperactivity behind poor regeneration in diabetic neuropathy?

Neuropathy is a troublesome complication of diabetes mellitus that commonly affects the sensory and autonomic nervous systems. Impaired nerve conduction and axonopathy lead to pain and/or paraesthesia followed by sensory loss. There is no effective treatment for diabetic neuropathy, in part because its mechanisms are poorly understood. Research has focused on correcting the changes induced by hyperglycaemia, such as oxidative stress, glycation of macromolecules, and mitochondrial dysfunction (Tomlinson and Gardiner, 2008). Neuroprotective strategies that target neurons, Schwann cells, and nerve blood supply have also been suggested (Calcutt et al. , 2008) but none have been successful (Apfel, 2002; Ropper et al. , 2009). However, in this issue of Brain , Douglas Zochodne and colleagues present evidence that inhibition of the phosphatase PTEN (phosphatase and tensin homolog) can produce functional improvements in a mouse model of diabetic neuropathy (Singh et al. , 2014). A frequent consequence of diabetic neuropathy, in addition to nerve degeneration, is the failure of axons to regenerate after injury. Recent studies of axon regeneration in the CNS have focused on the phosphatase PTEN. PTEN dephosphorylates the signalling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3) at position three. PIP3 mediates growth factor receptor signalling (including that of insulin) through activation of PI3-kinase at the plasma membrane. Activation of PTEN thus reduces activity in critical signalling pathways …

Clinical outcomes and survival of patients (pts) with sarcomatoid metastatic renal cell carcinoma (smRCC).

535 Background: SmRCC is a rare variant of RCC associated with poor outcome. No definitive therapeutic recommendations exist. We present survival outcomes in pts with smRCC treated with systemic therapy. Methods: From a single institutional database (years 2000-2012), we identified 21 pts with documented sarcomatoid features who received systemic therapy. Survival was assessed in the overall cohort and in subgroups divided by clinicopathologic characteristics, including the extent of sarcomatoid features, and Memorial Sloan-Kettering Cancer Center (MSKCC) risk criteria. Sarcomatoid component of the tumor was classified by predominant (≥20%) and non-predominant (<20%). Immunohistochemistry (IHC) analysis was performed for mammalian target of rapamycin (mTOR) signaling, phosphorylated ribosomal protein S6, proviral integration site proteins (PIM 1,2,3), Beta-catenin, E-cadherin, phosphatase and tensin homolog (PTEN), U3 small nucleolar ribonucleoprotein (IMP-3), p53, and epithelial membrane antigen (EMA) on tumor samples, where available. Results: 21 pts were included with a median age of 62 years (range 39-74) and 13 pts were male (62%). One pt received first line cytotoxic chemotherapy, 3 pts received first line immunotherapy, and 17 pts received targeted therapy. Pts belonged to the following MSKCC risk categories: 1 (5%) favorable, 13 (62%) intermediate and 7 (33%) poor risk. Median overall survival (OS) of the entire cohort was 249 days (95% CI 161-519 days). Median OS in pts stratified as MSKCC poor, intermediate, and good risk was 2556, 430, and 137 days respectively (Poor vs Intermediate HR 4.38, p = 0.0159, CI 1.30-19.90). There was no significant difference in OS in pts with sarcomatoid predominant disease vs. non-predominant disease (234 vs 286 days, p = 0.744, HR 0.85, CI 0.31-2.39). Conclusions: Overall OS in pts with smRCC is inferior compared to that reported in clear cell variant mRCC. Although MSKCC risk scores may be useful in determining prognosis, further studies are needed to identify relevant biomarkers and define the optimal therapeutic strategy for this disease. Correlation of survival outcomes with the IHC profile of tumors will be presented.