Tumor Suppressor Protein PTEN Research Articles

Transduction of PTEN Proteins Using the Tat Domain Modulates TGF-β1–Mediated Signaling Pathways and Transdifferentiation in Subconjunctival Fibroblasts

This study investigated the effects of the tumor suppressor protein PTEN (phosphatase and tensin homolog) on transforming growth factor (TGF)-β1-mediated signaling pathways and the transdifferentiation of human subconjunctival fibroblasts (SCFs) after the transduction of this protein containing a transactivator of transcription (Tat) domain. The Tat-PTEN expression vector was constructed to express the Tat domain of HIV-1 fused to PTEN. After transduction of the fusion protein and TGF-β1 stimulation, the dose-dependent effect of the transduced Tat-PTEN fusion protein on Akt phosphorylation and the stability of the Tat-PTEN fusion protein in SCF cells were evaluated by Western blot analysis. The effect of the Tat-PTEN fusion protein on the TGF-β1-stimulated expression of α-SMA and fibronectin was also evaluated by Western blot analysis and immunocytochemistry. To increase the efficiency of enzyme activity and to successfully deliver this protein to cells, the authors used a PTEN fusion protein that contained the transduction domain of the Tat protein from HIV-1. By Western blot analysis, the transduced Tat-PTEN fusion protein was found to modulate TGF-β1 signaling in SCF cells and result in the suppression of Akt phosphorylation. Furthermore, the transduction of the Tat-PTEN fusion protein was found to suppress the TGF-β1-stimulated expression of α-SMA and fibronectin by Western blot analysis and immunocytochemical staining, and the effects of the transduced fusion protein could be controlled in a dose-dependent manner. The Tat-PTEN fusion proteins were successfully transduced into the SCF cells and induced the suppression of transdifferentiation and fibrosis through the regulation of TGF-β-mediated signaling. The ability of the Tat-PTEN fusion protein to regulate cell survival could potentially be applied to protein therapy to counteract postoperative scarring in glaucoma surgery.

S-Nitrosylation of Mixed Lineage Kinase 3 Contributes to Its Activation after Cerebral Ischemia

Previous studies in our laboratory have shown that mixed lineage kinase 3 (MLK3) can be activated following global ischemia. In addition, other laboratories have reported that the activation of MLK3 may be linked to the accumulation of free radicals. However, the mechanism of MLK3 activation remains incompletely understood. We report here that MLK3, overexpressed in HEK293 cells, is S-nitrosylated (forming SNO-MLK3) via a reaction with S-nitrosoglutathione, an exogenous nitric oxide (NO) donor, at one critical cysteine residue (Cys-688). We further show that the S-nitrosylation of MLK3 contributes to its dimerization and activation. We also investigated whether the activation of MLK3 is associated with S-nitrosylation following rat brain ischemia/reperfusion. Our results show that the administration of 7-nitroindazole, an inhibitor of neuronal NO synthase (nNOS), or nNOS antisense oligodeoxynucleotides diminished the S-nitrosylation of MLK3 and inhibited its activation induced by cerebral ischemia/reperfusion. In contrast, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (an inhibitor of inducible NO synthase) or nNOS missense oligodeoxynucleotides did not affect the S-nitrosylation of MLK3. In addition, treatment with sodium nitroprusside (an exogenous NO donor) and S-nitrosoglutathione or MK801, an antagonist of the N-methyl-D-aspartate receptor, also diminished the S-nitrosylation and activation of MLK3 induced by cerebral ischemia/reperfusion. The activation of MLK3 facilitated its downstream protein kinase kinase 4/7 (MKK4/7)-JNK signaling module and both nuclear and non-nuclear apoptosis pathways. These data suggest that the activation of MLK3 during the early stages of ischemia/reperfusion is modulated by S-nitrosylation and provides a potential new approach for stroke therapy whereby the post-translational modification machinery is targeted.

Abstract B41: A mouse model for inflammation-driven lung tumorigenesis

Abstract The link between chronic inflammation and cancer development has lately gained extensive epidemiological and experimental support, particularly in organs like liver, stomach, colon, and lung. Although experimental models are available to study inflammation-driven tumorigenesis in the liver, stomach, and colon, no such models are available for lung cancer. Therefore, the objective of the present study is to develop a model for inflammation-driven lung tumorigenesis. A/J mice received four intraperitoneal injections (two doses/week) of the tobacco smoke carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at a dose level of 50 mg/kg body weight. Beginning one week after the last dose of NNK, the mice were given weekly intranasal instillations of lipopolysaccharide (LPS, 10ug/mouse), a potent inflammation-inducing agent, until the end of the study at week 27. Upon termination of the study, tumors on the surface of the lung were counted, their size determined and histopathologically analyzed. Moreover, we analyzed alterations in the expression of inflammation- and tumorigenesis-related proteins and microRNAs (miRs) by Western immunoblotting and qRT-PCR, respectively. Chronic exposure NNK-treated mice to LPS increased the number and size of lung tumors (47.3 ± 11 tumors/mouse for the NNK plus LPS group versus 28 ± 8 tumors/mouse in the NNK only group). Furthermore, lung tumors in the NNK plus LPS treated mice were significantly larger than the lung tumors of the mice challenged with NNK alone. Multiplicities of lung tumors in LPS-treated mice were similar to those mice given the vehicle. Multiplicities of the histopathological lesions pulmonary hyperplastic foci, adenoma, adenoma with dysplasia and adenocarcinoma in the NNK only group were 3.0 ± 1.2, 7.1 ± 2.9, 1.3 ± 1.1, and 0.3 ± 0.5/mouse, respectively. The corresponding frequencies of the lesions in the NNK plus LPS group were 3.1 ± 1.8, 11.8 ± 5.2, 3.9 ± 1.8, and 0.6 ± 0.7/mouse. Western immunoblotting studies showed that LPS exposure of NNK-treated mice enhanced activation of NF-kappaB, STAT3, and Akt but caused down-regulation of the tumor suppressor protein PTEN. Further, analyses of levels of five carcinogenesis-related microRNAs (miR-21, miR-146a, miR-150, miR-155 and miR-21, and miR-181b-1) showed increases in the expressions of miR-155 and miR-21. Levels of miR-155 and miR-21 were increased by 2.3-fold and 1.9-fold, respectively, in mice treated with NNK plus LPS group as compared to mice treated with NNK. Moreover, levels of miR-155 and miR-21 were increased by 10.5-fold and 4.7-fold, respectively, in LPS treated mice compared to vehicle-treated mice. In summary, our results demonstrate that LPS-induced chronic lung inflammation enhances lung tumorigenesis. This model could be a vey useful tool to understand the role inflammation in lung tumorigenesis and for identification of anti-inflammatory and lung cancer chemopreventive agents. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B41.

Correlation of HER2 and PTEN status with clinical outcome in esophageal (E), gastric (G), and gastroesophageal junction (GEJ) adenocarcinomas (ACs).

4066 Background: The determination of HER2 status is now critical for the management of E, G and GEJ AC's. Loss of expression of the PTEN tumor suppressor protein is considered to be a significant predictor of resistance to trastuzumab in the treatment of breast cancer. Methods: HER2 status was determined on 141 FFPE invasive AC's [13 E, 52 G and 76 GEJ] both by FISH (Abbott Molecular PathVysion) and IHC (Ventana Pathway and Dako HercepTeston a subset of cases) using the US FDA slide scoring criteria. PTEN expression was determined by IHC (rabbit mAB, Cell Signaling) using an automated method (Ventana) and semi-quantitative slide scoring. H&E slides were reviewed for all 44 endoscopic biopsies and 97 surgical resections included in the study, tumors were graded and staged according to the AJCC manual (7th ed). Results: While HER2 amplification/overexpression was markedly heterogeneous in both biopsy and resection specimens, extremely high correlation of FISH and IHC for HER2 status (p<0.001) was observed when the overexpressed foci were targeted for FISH examination. All (100%) IHC 2+ and 3+ cases were FISH+. Of 118 IHC 0 and 1+ cases, only 1 case (<1%) was discordant (FISH+). No cases featured HER2+ in situ carcinoma that did not also have HER2+ invasive carcinoma. HER2 status correlated with tumor site (26% GEJ vs. 8% E vs. 6% G, p=0.006), but not with grade, stage or survival. PTEN expression was lost in 70% of cases overall, and 83% (p=0.12) of HER2+ cases. PTEN loss was associated with advanced stage (p=0.005) and showed a trend toward correlation with shortened survival (p=0.09) in HER2+ cases. Conclusions: Unlike previous reports from the ToGA trial, in this series of 141 ACs there was extremely high correlation of FISH and IHC for determining HER2 status in E, G and GEJ tumors. HER2 is amplified/overexpressed in E, G and GEJ ACs, but not associated with aggressive disease in this study. PTEN expression was lost in the majority of HER2+ cases and correlated with aggressive disease. Further study of the association of PTEN with HER2 status and the interaction of PTEN with response to trastuzumab for the treatment of E, G and GEJ AC's appears warranted.

Activation of tumor suppressor protein PTEN and induction of apoptosis are involved in cAMP-mediated inhibition of cell number in B92 glial cells

During brain development, cAMP induces morphological changes and inhibits growth effects in several cell types. However, the molecular mechanisms underlying the growth inhibition remain unknown. Tumor suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that inhibits the phosphoinositide 3-kinase (PI3K) pathway. The phosphorylation of Akt, which is one of the key molecules downstream of PI3K, inhibits apoptosis. In this study, we investigated the role of PTEN in cAMP-mediated growth inhibition. B92 rat glial cells were treated with 2 different cAMP stimulatory agents, a phosphodiesterase (PDE) inhibitor and a β-adrenoceptor agonist. Both cAMP stimulatory agents induced marked morphological changes in the cells, decreased cell number, decreased Akt phosphorylation, activated PTEN, cleaved caspase-3, and induced the condensation and fragmentation of nuclei. These results indicate that the cAMP stimulatory agents induced apoptosis. Protein phosphatase inhibitor prevented cAMP-induced dephosphorylation of PTEN and Akt. In addition, cAMP analogs and Epac-selective agonists affected PTEN and Akt activities. These results suggested that cAMP-induced apoptosis may be mediated by PTEN activation and Akt inhibition through protein phosphatase in B92 cells. Our results provide new insight into the role of PTEN in cAMP-induced apoptosis in glial cells.

Abstract LB-132: Protein interactome between PTEN and cancer-associated mutant demonstrated the regulatory pathway of migration through its novel interactors

Abstract Mutation of the PTEN tumor suppressor protein frequently occurs in a variety of human cancers. PTEN is a phosphatase that negatively regulates the phosphatidyl-inositol-3-kinase (PI3K) pathway, and regulates cell proliferation, apoptosis, and cell motility. Though the tumor suppressive function involves PTEN's lipid phosphatase-dependent and -independent activities, the mechanism leading to the phosphatase-independent function of PTEN is poorly understood. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here we use a cancer associated mutant, G20E, to gain insight into the phosphatase-independent function of PTEN by investigating protein-protein interactions using mass spectrometry (MS)-based stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and SILAC has been developed to prioritize interactors and to compare the interactions between wild-type and G20E PTEN. Clustering of the prioritized interactors acquired by the PAP-SILAC approach are mainly involved in cell migration and apoptosis pathways, suggesting deregulation of the pathways could be involved in the tumorigenesis derived by the PTEN mutant. We further demonstrate that the one of wild-type specific interactor is required for the regulatory function of wild-type PTEN in cell migration. Furthermore, the other wild type specific interactor affects cell growth. These finding contribute to a better understanding of the mechanisms of PTEN's functions. 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 LB-132. doi:10.1158/1538-7445.AM2011-LB-132

Abstract 5371: A pooled apoptome shRNA screen to identify pathways that enhance NVP-BEZ235-induced apoptosis

Abstract NVP-BEZ235 is a dual inhibitor of class 1 phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) catalytic activity. It induces cell death in a subset of breast cancer cell lines characterized by amplification of human epidermal growth factor receptor 2 (HER2/ErbB2) and/or activating PIK3CA mutations, but not in cell lines with loss of function of the PTEN tumor suppressor protein or KRAS mutations. In order to better understand the molecular mechanisms leading to cell death, to identify potential biomarkers of activity and to reveal pathways that may even enhance NVP-BEZ235 induced lethality, a pooled short hairpin RNA (shRNA) screen using a lentiviral-based shRNA library targeting the apoptome was performed in NVP-BEZ235-sensitive MDA-MB453 and HCC1954 breast cancer cells. This strategy led to the identification of shRNAs that sensitized cells to NVP-BEZ235-induced apoptosis. Top sensitizers included pro-survival BCL2 family members, BCL2L1 and MCL1. Validation studies were carried out with single shRNAs targeting BCL2L1 and MCL1 as well as drug combination studies of NVP-BEZ235 with ABT-263 or ABT-747, both BCL2 homology domain 3 (BH3)-only protein mimetics. Cell death assays and biochemical readouts including PARP cleavage support the use of BCL2 family inhibitors to enhance NVP-BEZ235-induced cell death in HER2 amplified/PIK3CA mutated breast cancer cells. 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 5371. doi:10.1158/1538-7445.AM2011-5371

High glucose upregulation of early-onset Parkinson's disease protein DJ-1 integrates the PRAS40/TORC1 axis to mesangial cell hypertrophy

The Akt kinase signaling pathway is frequently deregulated in many human diseases including cancer, autoimmune disease and diabetes. In nephropathy, associated with diabetes, increased Akt signal transduction results in glomerular especially mesangial cell hypertrophy. The mechanism of Akt activation by elevated glucose is poorly understood. The oncogene DJ-1 prevents oxidative damage and apoptosis of dopaminergic neurons in animal models of Parkinson's disease and in culture. We identified DJ-1 to increase in response to high glucose in renal glomerular mesangial cells concomitant with an increase in phosphorylation of Akt in a time-dependent manner. Plasmid-derived overexpression as well as downregulation of DJ-1 by siRNA showed the requirement of this protein in high glucose-stimulated Akt phosphorylation. The tumor suppressor protein PTEN acts as a negative regulator of Akt activation. Interestingly, DJ-1 was associated with PTEN and this interaction was significantly increased in response to high glucose. High glucose-induced increase in DJ-1 promoted phosphorylation of the PRAS40, a negative regulator of TORC1 kinase activity, resulting in activating and inactivating phosphorylation of S6 kinase and 4EBP-1, respectively. Furthermore, DJ-1 increased protein synthesis and hypertrophy of mesangial cells. Our results provide evidence for a unique mechanism whereby DJ-1 induces Akt/PRAS40/TORC1-mediated hypertrophy in response to high glucose.

Abstract 965: Mechanistic studies of a dietary combination in cancer prevention: The effects of manganese superoxide dismutase on tumor suppressor gene activation

Abstract Mechanistic Studies of a Dietary Combination in Cancer Prevention: The Effects of MnSOD on Tumor Suppressor Gene Activation Reactive oxygen species can act as signaling transduction molecules modulating various pathways, such as the PI3K/Akt pathway. It is known that this pathway is constitutively activated in tumorigenesis contributing to protein synthesis, cell cycle progression, migration and survival. However, the PI3K/Akt pathway is negatively regulated by the major tumor suppressor protein PTEN. PTEN has dual specificity phosphatase activity regulating Akt phosphorylation as a lipid phosphatase; as well as, regulating cyclin D1 expression as a protein phosphotase. However, these are the roles of cytoplasmic PTEN. Recently, the nuclear roles of PTEN have received significant attention. Nuclear PTEN plays a major role in maintaining cellular homeostasis and stability by contributing to chromosome stability, DNA repair and cell cycle arrest. Previous studies have shown that PTEN nuclear localization is mediated by oxidative stress and may play a role in p53 stabilization. Nevertheless, MnSOD is considered to be a novel tumor suppressor gene. MnSOD has been defined as a nuclear encoded primary antioxidant enzyme localized in the mitochondria. We have shown in previous studies that the dietary combination Protandim reduced tumor incidence and multiplicity via the synergistic induction of superoxide dismutase enzymes. It has been shown that overexpression of MnSOD in in vivo models can suppress tumor formation via modulation of AP-1 binding. Therefore, it is imperative to look at how MnSOD may affect the process of apoptosis and the activation of tumor suppressor genes involved (i.e. PTEN and p53). Using our in vivo models (wildtype, MnSOD knockout, non-transgenic and MnSOD transgenic mice), we investigated how modulation of MnSOD expression may affect PTEN-mediated p53 stabilization, and how this contributes to chemoprevention. In addition, we explored how mutations in p53 may affect the PTEN-p53 interaction and its effects on tumor promotion via a gene transfection approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 965.

Response to erlotinib in recurrent glioblastoma multiforme showing coexpression of EGFRvIII and PTEN

Glioblastoma multiforme (GBM) is the most aggressive brain tumour in adults and remains incurable despite multimodal intensive treatment regimens. We present a patient with a recurrent glioblastoma who showed coexpression of the epidermal growth factor receptor mutant variant III (EGFRvIII) and the tumour-suppressor protein PTEN. She was treated with the tyrosine kinase inhibitor erlotinib for four months, achieving a partial response with improvement of neurologic symptoms. A review of the pertinent literature supporting the future use of therapies against epidermal growth factor receptor (EGFR) in highgrade gliomas is also provided.

A putative role for microRNA-205 in mammary epithelial cell progenitors

In an effort to understand the potential role of microRNAs (miRNAs) in mammary-gland stem or progenitor cells, miRNA microarrays were performed on subpopulations of the mouse mammary epithelial cell (MEC) line COMMA-DβGeo. This cell line contains a heterogeneous subpopulation of progenitors characterized by the expression of stem cell antigen 1 (Sca-1; encoded by Ly6a). Microarray analysis indicated that the Sca-1 subpopulations have distinct miRNA expression profiles. Functional studies were performed on miR-205, which was highly expressed in the Sca-1-positive (Sca-1+) cells. When miR-205 was overexpressed in vitro, the COMMA-DβGeo cells underwent several significant morphological and molecular changes. miR-205 overexpression led to an expansion of the progenitor-cell population, decreased cell size and increased cellular proliferation. In addition, the colony-forming potential of the two Sca-1 subpopulations was increased. Target prediction for miR-205 indicated that it might regulate the expression of the tumor-suppressor protein PTEN. Overexpression studies using reporter constructs confirmed that PTEN expression is regulated by miR-205. In addition to PTEN, several other putative and previously validated miR-205 targets were identified by microarray analysis, including the previously reported miR-205 targets ZEB1 and ZEB2. Additionally, in normal mouse MECs, high expression of miR-205 was observed in stem-cell-enriched cell populations isolated by FACS using established cell-surface markers.

A putative role for microRNA-205 in mammary epithelial cell progenitors.

In an effort to understand the potential role of microRNAs (miRNAs) in mammary-gland stem or progenitor cells, miRNA microarrays were performed on subpopulations of the mouse mammary epithelial cell (MEC) line COMMA-DbetaGeo. This cell line contains a heterogeneous subpopulation of progenitors characterized by the expression of stem cell antigen 1 (Sca-1; encoded by Ly6a). Microarray analysis indicated that the Sca-1 subpopulations have distinct miRNA expression profiles. Functional studies were performed on miR-205, which was highly expressed in the Sca-1-positive (Sca-1(+)) cells. When miR-205 was overexpressed in vitro, the COMMA-DbetaGeo cells underwent several significant morphological and molecular changes. miR-205 overexpression led to an expansion of the progenitor-cell population, decreased cell size and increased cellular proliferation. In addition, the colony-forming potential of the two Sca-1 subpopulations was increased. Target prediction for miR-205 indicated that it might regulate the expression of the tumor-suppressor protein PTEN. Overexpression studies using reporter constructs confirmed that PTEN expression is regulated by miR-205. In addition to PTEN, several other putative and previously validated miR-205 targets were identified by microarray analysis, including the previously reported miR-205 targets ZEB1 and ZEB2. Additionally, in normal mouse MECs, high expression of miR-205 was observed in stem-cell-enriched cell populations isolated by FACS using established cell-surface markers.

Simvastatin induces derepression of PTEN expression via NFκB to inhibit breast cancer cell growth

Sustained activation of Akt kinase acts as a focal regulator to increase cell growth and survival, which causes tumorigenesis including breast cancer. Statins, potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, display anticancer activity. The molecular mechanisms by which statins block cancer cell growth are poorly understood. We demonstrate that in the tumors derived from MDA-MB-231 human breast cancer cell xenografts, simvastatin significantly inhibited phosphorylation of Akt with concomitant attenuation of the expression of the anti-apoptotic protein Bcl XL. In many cancer cells, Bcl XL is a target of NFκB. Simvastatin inhibited the DNA binding and transcriptional activities of NFκB resulting in marked reduction in transcription of Bcl XL. Signals transmitted by anti-neoplastic mechanism implanted in the cancer cells serve to obstruct the initial outgrowth of tumors. One such mechanism represents the action of the tumor suppressor protein PTEN, which negatively regulates Akt kinase activity. We provide the first evidence for significantly increased levels of PTEN in the tumors of simvastatin-administered mice. Importantly, simvastatin markedly prevented binding of NFκB to the two canonical recognition elements, NFRE-1 and NFRE-2 present in the PTEN promoter. Contrary to the transcriptional suppression of Bcl XL, simvastatin significantly increased the transcription of PTEN. Furthermore, expression of NFκB p65 subunit inhibited transcription of PTEN, resulting in reduced protein expression, which leads to enhanced phosphorylation of Akt. Taken together, our data present a novel bifaceted mechanism where simvastatin acts on a nodal transcription factor NFκB, which attenuates the expression of anti-apoptotic Bcl XL and simultaneously derepresses the expression of anti-proliferative/proapoptotic tumor suppressor PTEN to prevent breast cancer cell growth.

PTEN and Ci-VSP Show Similar Phosphoinositide Binding Preferences

The Ciona intestinalis voltage sensor containing phosphatase (Ci-VSP) is a voltage dependent phosphatidylinositol phosphatase with two main domains, the voltage sensing domain (VSD) and the phosphatase domain (PD). Ci-VSP's PD bears homology with the phosphatase domain of the tumor suppressor protein PTEN (phosphatase and tensin homolog deleted chromosome 10). Recently, it has been proposed that the linker between the VSD and PD Ci-VSP constitute a Phosphoinositide Binding Motif (PBM). The PBM of Ci-VSP shares high homology with PTEN's N-terminal, which, in turn, is known to bind PI(4,5)P2 leading to an allosteric activation of PTEN. Similarly to the PBM of PTEN, we have found that a peptide representing the Ci-VSP's PBM (Ci-VSP240-271) binds PI(4,5)P2 significantly more strongly than other phosphatidyinositol bisphosphates. A Ci-VSP chimera created by replacing Arg 257 and Lys 258 with the amino acids found at the corresponding positions in the PTEN sequence, Gln 16 and Glu17, lead to a peptide that showed significantly reduced binding to PI(4,5)P2. While Ci-VSP240-271 exhibited a mixture of random and a-helical secondary structural elements, it was found that the chimeric Ci-VSP showed an increased b-sheet content. Molecular dynamics simulations were performed using the package NAMD and showed that the peptide does not form a helical structure and its charged residues interact in a pairwise fashion with PI(4,5)P2. Those pairs are form by R245 and R246, K252 and R253, and R254 and R257. Experimentally, the binding of the PBM has a great influence on the rate of return of the sensing charges. However, in a deletion mutant lacking the PD we observed that the PBM alone is not enough to limit the rate of return of the sensing currents. These observations strongly suggest that the binding of the PBM might be stabilized by the PD.

PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells

Backgroundc-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated.MethodsExpression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-γ agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migrationResultsHigh c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation.Conclusionc-Met is highly expressed in most tumors from patients with advanced-stage, metastatic NBL. Furthermore, using the NBL cell line SH-EP as a model, PHA665752 was shown to inhibit cMet/HGF/SF signaling in vitro, suggesting c-Met inhibitors may have efficacy for blocking local progression and/or metastatic spread of c-Met-positive NBL in vivo. These are novel findings for this disease and suggest that further studies of agents targeting the c-Met/HGF axis in NBL are warranted

Inpp5f Is a Polyphosphoinositide Phosphatase That Regulates Cardiac Hypertrophic Responsiveness

Cardiac hypertrophy occurs in response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has previously been strongly associated with hypertrophic signaling in the heart, and with the control of cell size in multiple contexts. This pathway is tightly regulated by many factors, including a host of kinases and phosphatases that function at multiple steps in the signaling cascade. For example, the PTEN (phosphatase and tensin homolog) tumor suppressor protein is a phosphoinositide 3-phosphatase that, by metabolizing phosphatidylinositol 3,4,5-trisphosphate (PtdIns[3,4,5]P(3), PIP3), acts in direct antagonism to growth factor-stimulated PI3K. Inhibition of PTEN leads to cardiomyocyte hypertrophy. Another polyphoinositide phosphatase, inositol polyphosphate-5-phosphatase F (Inpp5f) has recently been implicated in regulation of cardiac hypertrophy. Like PTEN, this phosphatase can degrade PtdIns(3,4,5)P(3) and thus modulates the PI3K/Akt pathway. To characterize the role of Inpp5f in regulating cardiac hypertrophy. We generated homozygous Inpp5f knockout mice and cardiac specific Inpp5f overexpression transgenic mice. We evaluated their hearts for biochemical, structural and functional changes. Inpp5f knockout mice have augmented hypertrophy and reactivation of the fetal gene program in response to stress when compared to wild-type littermates. Furthermore, cardiac overexpression of Inpp5f in transgenic mice reduces hypertrophic responsiveness. Our results suggest that Inpp5f is a functionally important endogenous modulator of cardiac myocyte size and of the cardiac response to stress.

Cholesteatoma growth and proliferation: posttranscriptional regulation by microRNA-21.

The goal of this study was to identify novel regulatory mechanisms controlling the growth and proliferation of cholesteatoma. Specifically, the potential role of microRNAs, regulators of protein translation, was studied in cholesteatoma. This study represents a molecular biologic investigation characterizing and comparing microRNA and protein expression in cholesteatoma and normal postauricular skin. Cholesteatoma and normal skin were taken from patients at the time of surgery. Tissue was processed for RNA and protein extraction. Real-time reverse-transcriptase-polymerase chain reaction was used to assess levels of human microRNAs, reverse-transcriptase-polymerase chain reaction was used to confirm the presence of upstream regulators, and Western blot analyses were used to assess levels of downstream target proteins. Among the microRNAs investigated, human microRNA-21 (hsa-miR-21) showed a 4.4-fold higher expression in cholesteatoma as compared with normal skin (p = 0.0011). The downstream targets of hsa-miR-21, PTEN and programmed cell death 4, were found to be greatly reduced in 3 of 4 cholesteatoma samples. Proposed upstream regulators of hsa-miR-21 expression (CD14, interleukin 6R, gp130, and signal transducer and activator of transcription 3) were present in all cholesteatoma tissues. MicroRNAs represent powerful regulators of protein translation, and their dysregulation has been implicated in many neoplastic diseases. This study specifically identified up-regulation of hsa-miR-21 concurrent with down-regulation of potent tumor suppressor proteins PTEN and programmed cell death 4. These proteins control aspects of apoptosis, proliferation, invasion, and migration. The results of this study were used to develop a model for cholesteatoma proliferation through microRNA dysregulation. This model can serve as a template for further study into potential RNA-based therapies for the treatment of cholesteatoma.

Neutral endopeptidase is a myristoylated protein

Neutral endopeptidase (NEP) is a cell-surface peptidase normally expressed by prostate epithelial cells and lost in ~50% of primary prostate cancers. NEP directly associates with multiple proteins at the cell surface including Ezrin/Radixin/Moesin (ERM) proteins and the PTEN tumor suppressor protein. Analysis of the N-terminal sequence of the NEP cytosolic domain (N-terminal MGKSESQMDI TDINTPKPKK KQRWTR) identified a myristoylation consensus site. Mutation of Gly-2 to Arg significantly decreased (3)H-myristoylation activity, and correlated with translocation of NEP from the plasma membrane to a perinuclear domain as demonstrated by immunofluorescence staining and Western blotting with an NEP-specific antibody. Removal of this myristoylation residue did not affect NEP enzymatic specific activity. Myristoylated NEP recruited more PTEN protein to the cell membrane fraction than unmyristoylated NEP. These data demonstrate that NEP is myristoylated at Gly-2 and that this modification is an intrinsic signal for membrane targeting.

Striking the balance between PTEN and PDK1: it all depends on the cell context: Figure 1.

The phosphatidyl-inosital-3 kinase (PI3K) signaling pathway is critical for normal brain development and function and is commonly hyperactivated in brain cancer. The PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor protein and phosphate-depended kinase 1 (PDK-1) are critical regulators of this pathway. In the July 15, 2009, issue of Genes & Development, Chalhoub and colleagues (pp. 1619-1624) demonstrate PDK1-dependent and PDK1-independent effects of conditional PTEN deletion in the brain, and they identify cell type-specific differences in feedback regulation of the PI3K pathway. These studies provide important insights as to how neurons and glia may differentially regulate PI3K signaling, yielding intriguing clues about targeting PTEN-deficient brain cancers.

5q– myelodysplastic syndromes: chromosome 5q genes direct a tumor-suppression network sensing actin dynamics

Complete loss or interstitial deletions of chromosome 5 are the most common karyotypic abnormality in myelodysplastic syndromes (MDSs). Isolated del(5q)/5q- MDS patients have a more favorable prognosis than those with additional karyotypic defects, who tend to develop myeloproliferative neoplasms (MPNs) and acute myeloid leukemia. The frequency of unbalanced chromosome 5 deletions has led to the idea that 5q harbors one or more tumor-suppressor genes that have fundamental roles in the growth control of hematopoietic stem/progenitor cells (HSCs/HPCs). Cytogenetic mapping of commonly deleted regions (CDRs) centered on 5q31 and 5q32 identified candidate tumor-suppressor genes, including the ribosomal subunit RPS14, the transcription factor Egr1/Krox20 and the cytoskeletal remodeling protein, alpha-catenin. Although each acts as a tumor suppressor, alone or in combination, no molecular mechanism accounts for how defects in individual 5q candidates may act as a lesion driving MDS or contributing to malignant progression in MPN. One candidate gene that resides between the conventional del(5q)/5q- MDS-associated CDRs is DIAPH1 (5q31.3). DIAPH1 encodes the mammalian Diaphanous-related formin, mDia1. mDia1 has critical roles in actin remodeling in cell division and in response to adhesive and migratory stimuli. This review examines evidence, with a focus on mouse gene-targeting experiments, that mDia1 acts as a node in a tumor-suppressor network that involves multiple 5q gene products. The network has the potential to sense dynamic changes in actin assembly. At the root of the network is a transcriptional response mechanism mediated by the MADS-box transcription factor, serum response factor (SRF), its actin-binding myocardin family coactivator, MAL, and the SRF-target 5q gene, EGR1, which regulate the expression of PTEN and p53-family tumor-suppressor proteins. We hypothesize that the network provides a homeostatic mechanism balancing HPC/HSC growth control and differentiation decisions in response to microenvironment and other external stimuli.