Tumor Suppressor Phosphatase Research Articles

DJ-1-induced phosphatase and tensin homologue downregulation is associated with proliferative and invasive activity of laryngeal cancer cells.

DJ-1, a novel mitogen-dependent oncogene, has an important role in the progression of human malignancies, whereas tumor suppressor phosphatase and tensin homolog (PTEN) is known to control a variety of processes associated with cell survival, proliferation and invasion. DJ-1 overexpression was reported to be negatively correlated with PTEN expression in tumor tissues of patients with laryngeal squamous cell carcinoma (LSCC). In the present study, the effect of DJ-1 on PTEN in laryngeal cancer cells was investigated by transfecting DJ-1-specific small interfering (si)RNA into Hep-2 and SNU-899 cells. Cell survival and cell proliferative and invasive capacity were then evaluated. The results showed that siRNA targeting of DJ-1 effectively upregulated PTEN expression, resulting in enhanced cell death as well as decreased proliferation and invasion of Hep-2 and SNU-899 cells. The results of the present study indicated, for the first time, to the best of our knowledge, that DJ-1-induced PTEN downregulation is associated with proliferative and invasive activity of laryngeal cancer cells. The DJ-1 gene may have an important role in the tumorigenesis of LSCC.

Lung neuroendocrine tumors: correlation of ubiquitinylation and sumoylation with nucleo-cytosolic partitioning of PTEN.

BackgroundThe tumor suppressor phosphatase and tensin homolog (PTEN) is a pleiotropic enzyme, inhibiting phosphatidyl-inositol-3 kinase (PI3K) signaling in the cytosol and stabilizing the genome in the nucleus. Nucleo-cytosolic partitioning is dependent on the post-translational modifications ubiquitinylation and sumoylation. This cellular compartmentalization of PTEN was investigated in lung neuroendocrine tumors (lung NET).MethodsTumor tissues from 192 lung NET patients (surgical specimens = 183, autopsies = 9) were investigated on tissue microarrays. PTEN was H-scored by two investigators in nucleus and cytosol using the monoclonal antibody 6H2.1. Results were correlated with immunoreactivity for USP7 (herpes virus-associated ubiquitin-specific protease 7) and SUMO2/3 (small ubiquitin-related modifier protein 2/3) as well as PTEN and p53 FISH gene status. Clinico-pathologic data including overall survival, proliferation rate and diagnostic markers (synaptophysin, chromogranin A, Mib-1, TTF-1) were recorded.ResultsThe multicentre cohort included 58 typical carcinoids (TC), 42 atypical carcinoids (AC), 32 large cell neuroendocrine carcinomas (LCNEC) and 60 small cell lung carcinomas (SCLC). Carcinoids were smaller in size and had higher synaptophysin and chromogranin A, but lower TTF-1 expressions. Patients with carcinoids were predominantly female and 10 years younger than patients with LCNEC/SCLC. In comparison to the carcinoids, LCNEC/SCLC tumors presented a stronger loss of nuclear and cytosolic PTEN associated with a loss of PTEN and p53. Concomitantly, a loss of nuclear USP7 but increase of nuclear and cytosolic SUMO2/3 was found. Loss of nuclear and cytosolic PTEN, loss of nuclear USP7 and increase of cytosolic SUMO2/3 thus correlated with poor survival. Among carcinoids, loss of cytosolic PTEN was predominantly found in TTF1-negative larger tumors of male patients. Among SCLC, loss of both cytosolic and nuclear PTEN but not proliferation rate or tumor size delineated a subgroup with poorer survival (all p-values <0.05).ConclusionsCellular ubiquitinylation and sumoylation likely influence the functional PTEN loss in high grade lung NET. Both nuclear and cytosolic PTEN immunoreactivity should be considered for correlation with clinico-pathologic parameters.

Catalysis by the tumor-suppressor enzymes PTEN and PTEN-L.

Phosphatase and tensin homologue deleted from chromosome ten (PTEN) is a lipid phosphatase tumor suppressor that is lost or inactivated in most human tumors. The enzyme catalyzes the hydrolysis of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) to form phosphatidylinositol-(4,5)-bisphosphate (PIP2) and inorganic phosphate. Here, we report on the first continuous assay for the catalytic activity of PTEN. Using this assay, we demonstrate that human PTEN is activated by the reaction product PIP2, as well as in solutions of low salt concentration. This activation is abrogated in the K13A variant, which has a disruption in a putative binding site for PIP2. We also demonstrate that PTEN-L, which derives from alternative translation of the PTEN mRNA, is activated constitutively. These findings have implications for catalysis by PTEN in physiological environments and could expedite the development of PTEN-based chemotherapeutic agents.

PTEN inhibition and axon regeneration and neural repair.

The intrinsic growth ability of all the neurons declines during development although some may grow better than others. Numerous intracellular signaling proteins and transcription factors have been shown to regulate the intrinsic growth capacity in mature neurons. Among them, PI3 kinase/Akt pathway is important for controlling axon elongation. As a negative regulator of this pathway, the tumor suppressor phosphatase and tensin homolog (PTEN) appears critical to control the regenerative ability of young and adult neurons. This review will focus on recent research progress in axon regeneration and neural repair by PTEN inhibition and therapeutic potential of blocking this phosphatase for neurological disorders. Inhibition of PTEN by deletion in conditional knockout mice, knockdown by short-hairpin RNA, or blockade by pharmacological approaches, including administration of selective PTEN antagonist peptides, stimulates various degrees of axon regrowth in juvenile or adult rodents with central nervous system injuries. Importantly, post-injury PTEN suppression could enhance axonal growth and functional recovery in adult central nervous system after injury.

Cowden Syndrome: Case Report, Update and Proposed Diagnostic and Surveillance Routines.

Cowden syndrome (CS) is an infrequent autosomal dominant multisystem genodermatosis, generally involving the skin, oral mucosa, thyroid, breast and gastrointestinal tract. It is characterized by a late onset in the 2nd or 3rd decade of life, an extraordinary potential for malignant transformation, especially of breast and thyroid, and an identifiable germline mutation. In 80% cases, the human tumor suppressor gene, phosphatase and tensin homolog (PTEN) is mutated; mutations involving KILLIN, SDH B/D, PIK3CA and AKT1 genes account for the rest of the cases. Its clinical signs are not only the “essential pearls” for early and accurate diagnosis of CS but also help timely detection of neoplasia as they precede development of cancer by several years. We describe the first Indian and the third world report of polydactyly with CS, review this entity highlighting on recent clinical developments and emphasize on regular and thorough screening for prompt identification and management of the potentially malignant growths. We have also designed a baseline workup routine as well as a detailed screening program for these patients.

Dynamic Studies of the Tumour Suppressor Protien PTEN Binding to Membranes Composed PI(4,5)P2 and Various Anionic Lipids

The tumor suppressor gene, Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) encodes a protein that is involved in many human cancers. It binds to the plasma membrane to dephosphorylate the 3-position of phosphatidylinositol-3,4,5-trisphosphate producing phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2). PTEN is an agonist to PI 3-kinase, thereby inhibiting the PI3K/Akt signaling pathway and controlling cell proliferation and survival. PTEN membrane association depends strongly on the composition and lateral distribution of the lipids in the membrane. Using stopped flow kinetic and steady state fluorescence experiments, we characterize the different steps associated with PTEN membrane association and dissociation.We find that PTEN membrane association is governed by the nature of the anionic lipids in the model membrane and their lateral distribution. It has been found previously that PTEN interacts synergistically with phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). When comparing the kinetics of PTEN binding to PC/PS (30%) and PC/PI(4,5)P2 (5%) vesicles, we find a faster kon value and slower koff rate constant for the PI(4,5)P2 containing vesicles. When both PS and PI(4,5)P2 are present in the vesicles, we find the fastest association rate constants. In model membrane systems, PS and PI(4,5)P2 show only limited co-localization. Therefore, the question arises whether PS in biological membranes is the second binding partner, in addition to PI(4,5)P2, of PTEN. We hypothesize that phosphatidylinositol (PI), which forms domains with PI(4,5)P2, may replace PS in at least some physiological scenarios as the second binding partner. We find that PTEN binding to PI vesicles is as strong as PTEN binding to PS vesicles, confirming the hypothesis that PTEN binding to PS or PI is non-specific electrostatic. This finding is a major paradigm shift in the PTEN field.

Phospholipid-binding Sites of Phosphatase and Tensin Homolog (PTEN): EXPLORING THE MECHANISM OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE ACTIVATION

The lipid phosphatase activity of the tumor suppressor phosphatase and tensin homolog (PTEN) is enhanced by the presence of its biological product, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This enhancement is suggested to occur via the product binding to the N-terminal region of the protein. PTEN effects on short-chain phosphoinositide (31)P linewidths and on the full field dependence of the spin-lattice relaxation rate (measured by high resolution field cycling (31)P NMR using spin-labeled protein) are combined with enzyme kinetics with the same short-chain phospholipids to characterize where PI(4,5)P2 binds on the protein. The results are used to model a discrete site for a PI(4,5)P2 molecule close to, but distinct from, the active site of PTEN. This PI(4,5)P2 site uses Arg-47 and Lys-13 as phosphate ligands, explaining why PTEN R47G and K13E can no longer be activated by that phosphoinositide. Placing a PI(4,5)P2 near the substrate site allows for proper orientation of the enzyme on interfaces and should facilitate processive catalysis.

PTEN expression in endothelial cells is down-regulated by uPAR to promote angiogenesis

The tumour suppressor phosphatase and tensin homologue (PTEN), mutated or lost in many human cancers, is a major regulator of angiogenesis. However, the cellular mechanism of PTEN regulation in endothelial cells so far remains elusive. Here, we characterise the urokinase receptor (uPAR, CD87) and its tumour-derived soluble form, suPAR, as a key molecule of regulating PTEN in endothelial cells. We observed uPAR-deficient endothelial cells to express enhanced PTEN mRNA- and protein levels. Consistently, uPAR expression in endogenous negative uPAR cells, down-regulated PTEN and activated the PI3K/Akt pathway. Additionally, we found that integrin adhesion receptors act as trans-membrane signaling partners for uPAR to repress PTEN transcription in a NF-κB-dependent manner. Functional in vitro assays with endothelial cells, derived from uPAR-deficient and PTEN heterozygous crossbred mice, demonstrated the impact of uPAR-dependent PTEN regulation on cell motility and survival. In an in vivo murine angiogenesis model uPAR-deficient PTEN heterozygous animals increased the impaired angiogenic phenotype of uPAR knockout mice and were able to reverse the high invasive potential of PTEN heterozygots. Our data provide first evidence that endogenous as well as exogenous soluble uPAR down-regulated PTEN in endothelial cells to support angiogenesis. The uPAR-induced PTEN regulation might represent a novel target for drug interference, and may lead to the development of new therapeutic strategies in anti-angiogenic treatment.

WWP2 is overexpressed in human oral cancer, determining tumor size and poor prognosis in patients: downregulation of WWP2 inhibits the AKT signaling and tumor growth in mice.

The WW domain containing E3 ubiquitin protein ligase 2 (WWP2) encodes a member of the Nedd4 family of E3 ligases, which catalyzes the final step of the ubiquitination cascade. WWP2 is involved in tumoral growth with degradation of the tumor suppressor phosphatase and tensin homologue deleted on chromosome TEN (PTEN). However, little is known about the mechanisms and roles of WWP2 in human malignancies including oral squamous cell carcinomas (OSCCs). We found frequent WWP2 overexpression in all OSCC-derived cell lines examined that was associated with cellular growth by accelerating the cell cycle in the G1 phase via degradation of PTEN and activation of the PI3K/AKT signaling pathway. Our in vivo data of WWP2 silencing showed dramatic inhibition of tumoral growth with increased expression of PTEN. Our 104 primary OSCCs had significantly higher expression of WWP2 than their normal counterparts. Moreover, among the clinical variables analyzed, enhanced WWP2 expression was correlated with primary tumoral size and poor prognosis. These data suggested that WWP2 overexpression contributes to neoplastic promotion via the PTEN/PI3K/AKT pathway in OSCCs. WWP2 is likely to be a biomarker of tumoral progression and prognosis and a potential therapeutic target for development of anticancer drugs in OSCCs.

PTEN induces apoptosis and cavitation via HIF-2-dependent Bnip3 upregulation during epithelial lumen formation.

The tumor suppressor phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 and antagonizes the prosurvival PI3K-Akt pathway. Targeted deletion of PTEN in mice led to early embryonic lethality. To elucidate its role in embryonic epithelial morphogenesis and the underlying mechanisms, we used embryonic stem cell-derived embryoid body (EB), an epithelial cyst structurally similar to the periimplantation embryo. PTEN is upregulated during EB morphogenesis in parallel with apoptosis of core cells, which mediates EB cavitation. Genetic ablation of PTEN causes Akt overactivation, apoptosis resistance and cavitation blockade. However, rescue experiments using mutant PTEN and pharmacological inhibition of Akt suggest that the phosphatase activity of PTEN and Akt are not involved in apoptosis-mediated cavitation. Instead, hypoxia-induced upregulation of Bnip3, a proapoptotic BH3-only protein, mediates PTEN-dependent apoptosis and cavitation. PTEN inactivation inhibits hypoxia- and reactive oxygen species-induced Bnip3 elevation. Overexpression of Bnip3 in PTEN-null EBs rescues apoptosis of the core cells. Mechanistically, suppression of Bnip3 following PTEN loss is likely due to reduction of hypoxia-inducible factor-2α (HIF-2α) because forced expression of an oxygen-stable HIF-2α mutant rescues Bnip3 expression and apoptosis. Lastly, we show that HIF-2α is upregulated by PTEN at both transcriptional and posttranscriptional levels. Ablation of prolyl hydroxylase domain-containing protein 2 (PHD2) in normal EBs or inhibition of PHD activities in PTEN-null EBs stabilizes HIF-2α and induces Bnip3 and caspase-3 activation. Altogether, these results suggest that PTEN is required for apoptosis-mediated cavitation during epithelial morphogenesis by regulating the expression of HIF-2α and Bnip3.

Understanding the tumor suppressor PTEN in chronic alcoholism and hepatocellular carcinoma.

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a phosphatidylinositol (PtdIns) phosphatase that regulates Akt activation via PtdIns 3 kinase. Changes in PTEN expression and/or activity have been identified in a variety of chronic hepatocellular disorders including obesity, NAFLD, NASH, and alcoholism. In cancer biology, PTEN is frequently mutated or deleted in a wide variety of tumors. Mutations, decreased promoter activity, and decreased expression in PTEN are frequently identified in patients with hepatocellular carcinoma. While the majority of research on PTEN concerns obesity and NASH, PTEN clearly has a role in hepatic insulin sensitivity and in the development of steatosis during chronic alcoholism. Yet, in chronic alcoholics and HCC, very little is known concerning PTEN mutation/deletion or low PTEN expression. This review is focused on an overview of the current knowledge on molecular mechanisms of dysregulation of PTEN expression/activity in the liver and their relationship to development of ethanol-induced hepatocellular damage and cancer.

PTEN Protein Loss and Clinical Outcome from Castration-resistant Prostate Cancer Treated with Abiraterone Acetate

BackgroundLoss of the tumor suppressor phosphatase and tensin homolog (PTEN) occurs frequently in prostate cancers. Preclinical evidence suggests that activation of PI3K/AKT signaling through loss of PTEN can result in resistance to hormonal treatment in prostate cancer. ObjectiveTo explore the antitumor activity of abiraterone acetate (abiraterone) in castration-resistant prostate cancer (CRPC) patients with and without loss of PTEN protein expression. Design, setting, and participantsWe retrospectively identified patients who had received abiraterone and had hormone-sensitive prostate cancer (HSPC) and/or CRPC tissue available for PTEN immunohistochemical analysis. Outcome measurements and statistical analysisThe primary end point was overall survival from initiation of abiraterone treatment. Relationship with outcome was analyzed using multivariate Cox regression and log-rank analyses. Results and limitationsA total of 144 patients were identified who had received abiraterone post-docetaxel and had available tumor tissue. Overall, loss of PTEN expression was observed in 40% of patients. Matched HSPC and CRPC tumor biopsies were available for 41 patients. PTEN status in CRPC correlated with HSPC in 86% of cases. Loss of PTEN expression was associated with shorter median overall survival (14 vs 21 mo; hazard ratio [HR]: 1.75; 95% confidence interval [CI], 1.19–2.55; p=0.004) and shorter median duration of abiraterone treatment (24 vs 28 wk; HR: 1.6; 95% CI, 1.12–2.28; p=0.009). PTEN protein loss, high lactate dehydrogenase, and the presence of visceral metastases were identified as independent prognostic factors in multivariate analysis. ConclusionsOur results indicate that loss of PTEN expression was associated with worse survival and shorter time on abiraterone treatment. Further studies in larger and prospective cohorts are warranted. Patient summaryPTEN is a protein often lost in prostate cancer cells. In this study we evaluated if prostate cancers that lack this protein respond differently to treatment with abiraterone acetate. We demonstrated that the survival of patients with loss of PTEN is shorter than patients with normal PTEN expression.

PTEN hamartoma tumor syndrome: Clinical risk assessment and management protocol

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important phosphatase that counteracts one of the most critical cancer pathways: the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathways. Clinically, deregulation of PTEN function resulting in reduced PTEN expression and activity is implicated in human diseases. Cowden syndrome (CS) is an autosomal dominant disorder characterized by benign and malignant tumors. CS-related individual features occur commonly in the general population. Approximately 25% of patients diagnosed with CS have pathogenic germline PTEN mutations, which increase lifetime risks of breast, thyroid, uterine, renal and other cancers. PTEN testing and intensive cancer surveillance allow for early detection and treatment of these cancers for mutation positive patients and their relatives. In this review, we highlight our current knowledge of germline PTEN mutations in relation to human disease. We review current clinical diagnosis and management recommendations for PHTS including recent discoveries in understanding PTEN function regulation and how this can be exploited therapeutically.

Abstract 3342: The p110β isoform of PI3K modulates response to growth factor receptor signaling in breast cancer cells irrespective of PTEN status

Abstract The class 1a phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth, survival, and metabolism. These PI3Ks are heterodimeric lipid kinases composed of a p85 regulatory subunit and a p110 catalytic subunit (p110α, p110β, or p110δ). p110α and p110β play distinct roles in PI3K signaling in carcinoma cells. p110α is activated by growth factor receptor kinase signaling. In contrast, p110β was shown to play a role in insulin metabolic action, G protein-coupled receptor (GPCR) signaling, and oncogenic transformation. Cancer cells deficient in PTEN, the tumor suppressor phosphatase that antagonizes PI3K signaling, have been shown to be sensitized to pharmacological inhibitors of p110β. As a result, early clinical studies with p110β inhibitors are restricted to patients with PTEN-deficient cancers. However, analysis of data from the Genomics of Drug Sensitivity in Cancer database revealed that genetic lesions in PTEN or PIK3CA (encodes p110α) were significantly and independently associated with increased sensitivity to the p110β inhibitor AZD6284. Among 668 cancer cell lines, 96, 57, and 5 lines harbored alterations in PTEN, PIK3CA, or both, respectively. Among 61 cell lines with IC50 values ≤2 μM, only 25 harbored an alteration in PTEN and/or PIK3CA. Thus, a significant fraction of cancer cell lines (and tumors) without PTEN alterations are likely to be sensitive to p110β inhibition. To explore the role of p110β in PI3K signaling in ER+ breast cancer, we treated cells with the p110β inhibitor GSK2636771 and monitored effects on growth factor-induced activation of AKT and MEK/ERK. We found that p110β inhibition reduced basal pAKT and pERK levels in PTEN-deficient cells. Furthermore, p110β inhibition reduced insulin-like growth factor 1 (IGF-1)-induced pAKT levels and delayed the onset of AKT phosphorylation in both PTEN-deficient and PTEN-wild-type cells. In contrast, p110β inhibition sensitized both PTEN-wildtype and PTEN-deficient cells to heregulin stimulation. These results indicate that p110β desensitizes cells to IGF-1 stimulation and hyper-sensitizes them to heregulin stimulation and suggests that p110β regulates downstream AKT and MEK/ERK activation in response to growth factor receptor activation. Our findings advocate for the testing of p110β inhibitors in cell lines (and tumors) selected based on growth factor dependence and PTEN status. Citation Format: Stephanie J. Bouley, Lloye M. Dillon, Todd W. Miller. The p110β isoform of PI3K modulates response to growth factor receptor signaling in breast cancer cells irrespective of PTEN status. [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 3342. doi:10.1158/1538-7445.AM2014-3342

Abstract 5313: Phosphoproteomic analysis with reverse phase protein array identifies N-myc downstream regulated gene 1 (NDRG1) as a biomarker of phosphatidylinositol-3-kinase (PI3K)α inhibitor activity in a PTEN-deficient glioma cell line

Abstract Aberrant activation of phosphoinositide 3-kinase (PI3K) signaling is commonly reported in cancer, and leads to deregulation of several intracellular processes, including cell survival, growth, proliferation and migration. PI3K activates AKT, serum/glucocorticoid regulated kinase (SGK), phosphoinositide -dependent kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and several other molecules involved in cell progression and survival. Reports have also indicated that the deletion of the gene encoding the tumor suppressor phosphatase and tensin homologue (PTEN) is one of the key factors controlling activation of the PI3K pathway. Glioblastoma (GBM), the most common malignant brain tumor in adults, represents a compelling disease for kinase inhibitor therapy. Although many therapies to target the Ras-PI3K-mTOR axis in GBM have been attempted, their efficacies have not been pronounced, presumably because of the complexity of PI3K mediated signaling. To elucidate the role of PTEN in PI3K signaling, the phosphorylation status of two glioma cell lines, LN229 (wild type PTEN) and U-87 MG (PTEN deficient) was investigated with phosphoproteomic reverse phase protein array (RPPA). When basal phosphorylation was compared between both cell lines, the phosphorylation of N-myc downstream regulated gene 1 (NDRG1) (Thr346), a physiological substrate of SGK1, was increased in U-87 MG cells compared with levels in LN229 cells. These data suggest that PTEN deletion affects the phosphorylation status of NDRG1 in U-87 MG cells. Following treatment with PIK-90, a PI3Kα inhibitor, phosphorylation of Akt (Ser473) and its direct substrate PRAS40 (Thr246) were decreased in both cell lines, however, NDRG1 (Thr346) phosphorylation was strongly inhibited only in U-87 MG cells. These results suggest that the differential effects of PIK-90 on LN229 and U-87 MG cells is PTEN dependent, and that detecting NDRG1 (Thr346) phosphorylation could be a biomarker for PI3Kα inhibitor treatment in PTEN deleted cancer cells. Ongoing studies are focusing on elucidating the mechanism of action of PIK-90 in glioma cell lines. Citation Format: Takeomi Inoue, Naoko Iwata, Eiji Nishiwaki, Yasuyuki Kirii. Phosphoproteomic analysis with reverse phase protein array identifies N-myc downstream regulated gene 1 (NDRG1) as a biomarker of phosphatidylinositol-3-kinase (PI3K)α inhibitor activity in a PTEN-deficient glioma cell line. [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 5313. doi:10.1158/1538-7445.AM2014-5313

Analysis of PTEN ubiquitylation and SUMOylation using molecular traps

The function of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is tightly controlled by post-translational modifications (PTMs) including ubiquitin or Small Ubiquitin-related MOdifiers (SUMO). It is known that SUMOylation by SUMO-1, SUMO-2/-3, mono- or polyubiquitylation have a distinct impact on PTEN activity, localisation and/or stability, however the molecular mechanisms governing these processes are still unclear. Studying PTM regulated events has always been a difficult task due to their labile nature. Here, we propose an update on the role of these PTMs on PTEN function, as well as a methodological overview on the use of molecular traps named SUMO Binding Entities (SUBEs) or Tandem Ubiquitin Binding Entities (TUBEs) to capture SUMOylated or Ubiquitylated forms of PTEN respectively. When combined with in vitro SUMOylation or Ubiquitylation assays, the use of molecular traps facilitate the detection of modified forms of PTEN. SUMO and ubiquitin-traps are also suitable to capture endogenously modified forms of PTEN after expression of E3 ligases or treatment with chemical inhibitors. This versatile approach represents an interesting alternative to explore PTEN regulation by SUMO and ubiquitin under physiological or pathological conditions.

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.

Hesperidin induces apoptosis and triggers autophagic markers through inhibition of Aurora-A mediated phosphoinositide-3-kinase/Akt/mammalian target of rapamycin and glycogen synthase kinase-3 beta signalling cascades in experimental colon carcinogenesis

Abnormalities in the homeostasis mechanisms involved in cell survival and apoptosis are contributing factors for colon carcinogenesis. Interventions of these mechanisms by pharmacologically safer agents gain predominance in colon cancer prevention. We previously reported the chemopreventive efficacy of hesperidin against colon carcinogenesis. In the present study, we aimed at investigating the potential of hesperidin over the abrogated Aurora-A coupled pro-survival phosphoinositide-3-kinase (PI3K)/Akt signalling cascades. Further, the role of hesperidin over apoptosis and mammalian target of rapamycin (mTOR) mediated autophagic responses were studied. Azoxymethane (AOM) induced mouse model of colon carcinogenesis was involved in this study. Hesperidin treatment was provided either in initiation/post-initiation mode respectively. Hesperidin significantly altered AOM mediated anti-apoptotic scenario by modulating Bax/Bcl-2 ratio together with enhanced cytochrome-c release and caspase-3, 9 activations. In addition, hesperidin enhanced p53–p21 axis with concomitant decrease in cell cycle regulator. Hesperidin treatment caused significant up-regulation of tumour suppressor phosphatase and tensin homologue (PTEN) with a reduction in the expression of AOM mediated p-PI3K and p-Akt. Additionally, hesperidin administration exhibited inhibition against p-mTOR expression which in turn led to stimulation of autophagic markers Beclin-1 and LC3-II. Aurora-A an upstream regulator of PI3K/Akt pathway was significantly inhibited by hesperidin. Furthermore, hesperidin administration restored glycogen synthase kinase-3 beta (GSK-3β) activity which in turn prevented the accumulation of oncoproteins β-catenin, c-jun and c-myc. Taken together, hesperidin supplementation initiated apoptosis via targeted inhibition of constitutively activated Aurora-A mediated PI3K/Akt/GSK-3β and mTOR pathways coupled with autophagic stimulation against AOM induced colon carcinogenesis.

CIP2A and PP2A in human leptomeninges, arachnoid granulations and meningiomas

Previously we have found that mitogens stimulate proliferation of meningioma cells of all grades, in part, by activation of the PI3K-PKB/Akt-PRAS40-mTOR pathway regulated to some degree by the tumor suppressor phosphatase PP2A. PP2A activity is inhibited by the oncoprotein cancerous inhibitor of PP2A (CIP2A), which has not been studied in meningiomas to our knowledge. Six fetal and one adult human leptomeningeal samples and 38 meningiomas were evaluated by western blot. Fifteen adult arachnoid granulations and 58 formalin-fixed meningiomas (36 World Health Organization grade I, 15 grade II and seven grade III) were evaluated by immunohistochemistry. The effects of the mitogens platelet derived growth factor-BB (PDGF-BB) and cerebrospinal fluid on CIP2A were also studied. By western blot, CIP2A and PP2A were found in the five fetal and one adult leptomeninges and all meningiomas. By immunohistochemistry, CIP2A was detected in the arachnoid granulations and all meningiomas. CIP2A tended to be higher in grade III tumors. Three fetal leptomeningeal (two grade I and one grade II) and meningioma cells treated with PDGF-BB and/or human cerebrospinal fluid resulted in a slight increase in CIP2A in the leptomeningeal cells but not meningioma cells. Considered the mechanism of action and seen in other neoplasms, these findings raise the possibility that CIP2A may participate in the biology of meningiomas.

Glucose modulates Pax6 expression through the JNK/p38 MAP kinase pathway in pancreatic beta-cells

AimThe paired and homeodomain-containing transcription factor, paired box 6 (Pax6), has shown to play pivotal roles in beta-cell function, including cell survival, insulin biosynthesis and secretion. The present study investigates the signaling events that regulate the modulation of Pax6 expression by glucose and the role of this modulation in cell survival in rat insulinoma-1E (INS-1E) cells. Main methodsINS-1E cells were incubated on 1mM (low) or 25mM (high) glucose overnight. To elucidate the signaling pathways that regulate Pax6 expression, we utilized specific inhibitors. The siRNA transfection of Pax6 into INS-1E cells was performed by electroporation. The mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. Key findingsWe found that the mRNA and protein levels of Pax6 were reduced by approximately 4-fold in high, compared to low, glucose-treated cells. Staurosporine, the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 significantly increased Pax6 levels in high glucose-treated INS-1E cells compared to their respective controls. However, neither calcium ionophore nor the extracellular signal-regulated kinase (ERK) inhibitor U0126 resulted in any alteration in Pax6 protein expression. Further, a siRNA-mediated knockdown of Pax6 significantly decreased the expression of tumor-suppressor phosphatase with tensin homology (PTEN) while increasing cell viability in low glucose-treated INS-1E cells. SignificanceThis study addresses the signaling events that regulate the glucose-dependent expression of Pax6 and the role of these events in cell survival in pancreatic beta cells.