Deformed epidermal autoregulatory factor -1 (DEAF-1) is a transcription factor mapping to the chromosomal region 11p15.5, a region associated with loss of heterozygosity (LOH) in human cancers. Potential DEAF-1 binding motifs were identified in the PTEN promoter and the ability of DEAF-1 to regulate PTEN gene expression was investigated. DEAF-1 increased transcription 10-14 fold with PTEN sequences between -429 and -221, while mutations in the DNA binding domain (DEAF-ADWA) and nuclear localization signal of DEAF-1 abolished this increase. DEAF-1 was shown to bind sequences between -339 and -233. Rabdomyosarcoma (RD) stable cell lines with inducible expression of DEAF-1 and DEAF-1-ADWA were produced. Increased DEAF-1 expression had no significant effects on PTEN RNA expression or cell proliferation when compared to controls, but did increase susceptibility to UV-induced apoptosis. These studies suggest that DEAF-1 may contribute to the regulation of PTEN gene expression, but other factors may play a more significant role.

Submitted by FERES ABRÃO null (feresabrao@terra.com.br) on 2016-07-13T11:38:27Z No. of bitstreams: 1 Doutorado correção.pdf: 4863333 bytes, checksum: d5c66d377ec9c9a5af09337fc25585bd (MD5) / Rejected by Ana Paula Grisoto (grisotoana@reitoria.unesp.br), reason: Submissão duplicada. Solicitamos que realize uma nova submissão. Agradecemos a compreensão. on 2016-07-14T20:50:33Z (GMT) / Submitted by FERES ABRÃO null (feresabrao@terra.com.br) on 2016-07-14T21:08:33Z No. of bitstreams: 1 Doutorado correção.pdf: 4863333 bytes, checksum: d5c66d377ec9c9a5af09337fc25585bd (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-15T17:20:39Z (GMT) No. of bitstreams: 1 abrao_f_dr_bot.pdf: 4863333 bytes, checksum: d5c66d377ec9c9a5af09337fc25585bd (MD5) / Made available in DSpace on 2016-07-15T17:20:39Z (GMT). No. of bitstreams: 1 abrao_f_dr_bot.pdf: 4863333 bytes, checksum: d5c66d377ec9c9a5af09337fc25585bd (MD5) Previous issue date: 2016-02-19 / Objetivo: Avaliar os pólipos endometriais pela imunoexpressão das proteínas p53 e PTEN como fatores preditivos para riscos de pré-malignidade e malignidade. Pacientes e Métodos: Estudo transversal com amostra por conveniência, cujos dados foram obtidos através de consultas aos prontuários no período de janeiro de 2010 a dezembro 2014 de pacientes com diagnóstico de pólipos endometriais, submetidas a histeroscopias diagnósticas e cirúrgicas/polipectomia no Setor de Endoscopia Ginecológica e Planejamento Familiar da Disciplina de Ginecologia da Faculdade de Medicina de Botucatu (UNESP) e da Clínica “Abrão” de Marília. Os pólipos endometriais foram encaminhados, em bloco de parafina, à Consultoria em Patologia Dr Carlos Bacchi para avaliação imunoistoquímica, identificando os marcadores p53 e PTEN. Todas as pacientes realizaram, previamente, ultrassonografia transvaginal e histeroscopia diagnóstica. O grupo estudado constituiu-se de 159 pacientes (n=159) com diagnóstico de pólipo confirmado pelo exame anátomo patológico. Grupo amostral: as pacientes foram divididas em 2 (dois) grupos, assim denominados: Grupo A=120 (pacientes com pólipos endometriais sem atipias) e Grupo B=39 (pacientes com pólipos endometriais com atipias). O Grupo A foi subdividido em 4 subgrupos, assim denominados: A1- imunoistoquímico p53 negativo e PTEN positivo; A2- imunoistoquímico p53 positivo e PTEN positivo; A3- imunoistoquímico p53 negativo e PTEN negativo e A4- imunoistoquímico p53 positivo e PTEN negativo. O Grupo B foi subdividido em 4 subgrupos, assim denominados: B1- imunoistoquímico p53 negativo e PTEN positivo; B2- imunoistoquímico p53 positivo e PTEN positivo; B3- imunoistoquímico p53 negativo e PTEN negativo e B4 imunoistoquímico p53 positivo e PTEN negativo. Resultados: Os Grupos não apresentaram diferenças nas características clinico-epidemiologicas, demonstrando homogeneidade na amostra estudada. No grupo A: o subgrupo A1, com 80 pacientes (66,6%) apresentou p53 negativo e PTEN positivo, marcadores tumorais normais e 1 (uma) paciente (1,25%) teve neoplasia maligna de endométrio. As demais 40 pacientes (33,4%) dos subgrupos A2, A3, A4 apresentaram marcadores tumorais alterados, destes, 6 (seis) pacientes (15%) tiveram neoplasia maligna de endometrio; O A2 com 20 pacientes (16,6%) apresentou p53 positivo e PTEN positivo; A3 com 14 pacientes (11,6%) apresentou p53 positivo e PTEN negativo e A4 com 6 pacientes (5,1%) apresentou p53 negativo e PTEN negativo . O Grupo A teve, média de idade 57,5 anos, Índice de Massa Corpórea (IMC) 28,45, número médio de gestações 2(duas), índice de Aborto 16,7%, Tabagista 8,3%, Hipertensas 46,7%, Diabeticas 11,7%, amenorreia média 10 anos. Cerca de 5.9% das pacientes do grupo A tiveram neoplasia maligna de endométrio e houve maior incidência de neoplasia nas pacientes com marcadores alterados, em relação aquelas com marcadores normais (15% versus 1,25%, respectivamente; p=0,0089 e OR=13.94). No Grupo B: o subgrupo B1 com 21 pacientes (53,9%) apresentou marcadores tumorais normais (p53 negativo e PTEN positivo) e 1(uma) paciente (4,8%) teve neoplasia maligna de endométrio. As demais 18 pacientes (46,1%) dos subgrupos B2, B3, B4 apresentaram marcadores tumorais alterados, destes 7(sete) pacientes (38,9%) tiveram neoplasia maligna de endométrio. O B2 com 11 pacientes (28,2%) apresentou p53 positivo e PTEN positivo; B3 com 4 pacientes (10,2%) apresentou p53 positivo e PTEN negativo e A4 com 3 pacientes (7,7%) apresentou p53 negativo e PTEN negativo. O Grupo B teve, média de idade 61 anos, Índice de Massa Corpórea (IMC) 27, número médio de Gestações 2 (duas), índice de Aborto 7,7%, Tabagista 8,3%, Hipertensas 64%, Diabeticas 17,9%, com amenorreia média de 10 anos. Cerca de 20,5% das pacientes do grupo B tiveram neoplasia maligna de endométrio e houve maior incidência nas pacientes com marcadores tumorais alterados em relação as pacientes com marcadores normais (38,9 versus 4,8%, respectivamente; p=0,00255 e OR= 12.73). O grupo B teve maior incidência de neoplasia maligna de endometrio em relação ao grupo A (20,5% versus 5,9% respectivamente; p=0,011). No total 159 pacientes (grupo A + Grupo B), 110 pacientes com p53 negativo, 3 pacientes tiveram neoplasia de endométrio; e 49 pacientes com p53 positivo, 11 tiveram neoplasia de endométrio.(p=0,0006,OR=7,67) e 132 pacientes com PTEN positivo, 8 pacientes tiveram neoplasia de endométrio, 27 pacientes com PTEN negativo, 7 tiveram neoplasia de endométrio.(p=0,00043; OR=5,43). Conclusões: 1- O estudo imunoistoquímico nas pacientes portadoras de pólipo endometrial mostrou ser um armamento útil para predizer o risco de malignização do endométrio,2- Estudos complementares devem ser aguardados, principalmente com maior casuística, para corroborar os dados encontrados,3- As pacientes com pólipos endometriais e marcadores tumorais alterados, p53 e PTEN, principalmente, quando associados a obesidade, hipertensão arterial sistêmica e diabetes mellitus II, podem apresentar maior risco de neoplasia maligna de endométrio,4- As neoplasias malignas do endométrio foram encontrados com maior incidências em mulheres de maior idade e no grupo de pólipos com atipias. / Objective: Evaluate the endometrial polyps by immune expression of p53 protein and PTEN as predictive factors for pre malignancy and malignancy risks. Patients and Methods: Transversal study with samples by convenience whose data were obtained by consulting the medical charts from January 2010 to December 2014 of patients diagnosed with endometrial polyps, submitted to a diagnostic and surgical hysteroscopy/polypectomy in the Gynaecological Endoscopy and Familiar Planning Division of the Gynaecology Discipline at Medical School of Botucatu (UNESP) and “Abrão” Clinic of Marília. The endometrial polyps, in paraffin blocks, were sent to Doctor Carlos Bacchi Pathology Advisory for immune hystochemical assessment, identifying the markers p 53 and PTEN. Previously, transvaginal ultrasonography and diagnostic hysteroscopy were done in all the patients. The studied group was made up of 159 patients (n=159) with confirmed diagnosis of polyps by anatomical pathological examination. Sample group: the patients were divided into 2 groups: Group A=120 (patients with endometrial polyps without atypias) and Group B = 39 (patients with endometrial polyps with atypias). Group A was subdivided into 4 sub groups: A1- immune hystochemical p53 negative and PTEN positive; A2- immune hystochemical p53 positive and PTEN positive; A3- immune hystochemical p53 negative and PTEN negative and A4- immune hystochemical p53 positive and PTEN negative. Group B was subdivided into 4 sub groups: B1- immune hystochemical p53 negative and PTEN positive; B2- immune hystochemical p53 positive and PTEN positive; B3- immune hystochemical p53 negative and PTEN negative and B4- immune hystochemical p53 positive and PTEN negative. Results: The groups did not present differences in clinical-epidemiological characteristics, showing homogeneity in the sample studied. In Group A: subgroup A1 with 80 patients (66.6%) presented p53 negative and PTEN positive, normal tumor markers and one patient (1.25%) had endometrium malignant neoplasm. The other 40 patients (33.4%) of subgroups A2, A3 and A4 presented altered tumor markers, being 6 (six) patients (15%) with endometrium malignant neoplasm; A2 with 20 patients(16.6%) presented p53 positive and PTEN positive; A3 with 14 patients (11.6%) presented p 53 positive and PTEN negative and A4 with 6 patients (5.1 %) presented p53 negative and PTEN negative. In Group A, the average age was 57.5 years, the Body Mass Index (BMI) 28.45, the medium number of Pregnancy was 2(two), the Abortion rate was 16.7%, Smoking 8.3%, Hypertensive 46.7%, Diabetics 11.7%, with 10 years average amenorrhea, and around 5.9% of them developed endometrium malignant neoplasm and there was higher incidence of neoplasm on patients with altered markers compared to those with normal markers (15% versus 1.25%, respectively; p=0.0089 and OR=13.94). In Group B: the subgroup B1 with 21 patients (53.9%) presented normal tumor markers (p53 negative and PTEN positive) and 1(one) patient (4.8%) had endometrium malignant neoplasm. The other 18 patients (46.1%) of subgroups B2, B3, B4 presented altered tumor markers, being that 7(seven) patients (38.9%) had endometrium malignant neoplasm. B2 with 11 patients (28.2%) presented p53 positive and PTEN positive; B3 with 4 patients (10.2%) presented p53 positive and PTEN negative and A4 with 3 patients (7.7%) presented p53 negative and PTEN negative. In Group B, the average age was 61 years, the Body Mass Index (BMI) 27, the medium number of Pregnancy was 2 (two) , the Abortion rate was 7.7%, Smoking 8.3%, Hypertensive 64%, Diabetics 17.9%, with 10 years average amenorrhea, and around 20.5% of them developed endometrium malignant neoplasm and there was higher incidence on patients with altered tumor in relation to those with normal markers (38.9 versus 4.8%, respectively; p=0.00255 e OR= 12.73). Group B had higher incidence of endometrium malignant neoplasm than group A (20.5% versus 5.9% respectively; p=0.011). Out of 159 patients (Group A + Group B), 110 patients with p53 negative, 3 patients had endometrium neoplasm; and 49 patients with p53 positive, 11 had endometrium neoplasm.(p=0.0006,OR=7.67) and 132 patients with PTEN positive, 8 patients had endometrium neoplasm, 27 patients with PTEN negative, 7 had endometrium neoplasm.(p=0.00043; OR=5.43). Conclusions:1-Immunohistochemical analysis can be useful to predict malignant transformation in cases of endometrial polyps, 2-Further larger studies to confirm the data obtained are warranted, 3-The risk of malignant endometrial neoplasia is higher in patients with endometrial polyps showing abnormal p53 and PTEN immunohistochemistry in the presence of advanced age, high BMI, systemic arterial hypertension, and type 2 Diabetes Mellitus, 4-The incidence of malignant endometrial neoplasia was higher in women of more advance age with polyps with atypia.

L'inactivation des gènes p53 et pten présente un facteur de mauvais pronostic et de résistance à différents traitements anticancéreux incluant les thérapies ciblées. Le cétuximab (Erbitux®) est un anticorps monoclonal chimérique dirigé contre le domaine extracellulaire du récepteur à l'EGF. Son mécanisme d'action est fortement lié à la fonctionnalité des voies de signalisation de PI3KJAKT et des MAPK. Nous avons évalué l'influence de la réintroduction des gènes p53 et pten par vectorisation non viral à base de polyéthylènimine, couplée ou non à l'internalisation photochimique sur l'induction de l'apoptose et l'inhibition de la croissance cellulaire dans des cellules P53 et pten mutés d'une part, et sur l'impact de ce transfert de gène sur la fonctionnalité des voies de signalisation impliquées dans la réponse cellulaire au cétuximab. Nous avons montré que la transfection des gènes suppresseurs de tumeurs p53 et pten par polyétylènimine couplé à la PCI rétablit l'expression de P53 et de PTEN et permet de restaurer l'inhibition de croissance et l'induction de l'apoptose. Associée au traitement par cétuximab La restauration de PTEN et P53 réprime l'activation constitutive des voies de signalisation PI3K et MAPK et potentialise l'inhibition de la croissance et l'induction de l'apoptose induites par le cétuximab. Ces résultats montrent que l'inactivation de p53 et pten pourrait être prédictive de la résistance au cétuximab à travers l'activation constitutive de la voie de signalisation PI3/AKT et confirme que la détennination de la fonctionnalité des voies de signalisation des rtiveaux d'expression des phosphoprotéines de signalisation permettrait de prédire la réponse au cétuximab et de proposer des options thérapeutiques originales. / P53 and PTEN abnormalities are early events in carcinogenesis and are associated with poor prognosis and resistance to cancer therapies including targeted therapy. Cetuxirnab (Erbitnx®) is a IgG chimeric monoclonal antibody, directed against the extracellular dornain of EGFR. !ts activity has been shown to be dependent on the functionality of PI3KJAKT and MAPK signaling pathways as weil as the apoptosis induction capacity of the cells . The present study was designed to evaluate the influence of pten and p53 gene transfer using polyethylenimine with or without photochemical internalisation on cell apoptosis induction and cell growth in cells bearing p53 and pten mutations and on the response to cetuximab. The results presented show that pten and p53 gene transfer using polyethertimine and PCI restored P53 and PTEN expression, cell growth inhibition and apoptosis induction. Associated with cetuximab treatrnent, pten and p53 reintroduction, restored the functionality of AKTIPI3K and MAPK signaling pathways and increased cell growth inhibition and apoptosis induction by cetuximab. The results achieved in the present study show that pten and p53 mutations could be predictive of cell response to cetuximab through the functional impact of these mutations on cell signaling. The data presented put forward the interest of the analysis of EGFR phosphoproteins downstream signalling to evaluate the functionality of the signaling pathways implicated in the cell response to cetuximab and could be used to propose the original targeted therapies.

E-selectin is a transmembrane endothelium adhesion protein involved in rolling, arrest and migration of leukocytes as well as in the metastasis of many cancer types. Previous reports suggested that the interactions between E-selectin and its ligands transduce signals into migrating leukocytes and in E-selectin expressing endothelial cells. This study investigates the signaling pathways involved in E-selectin binding to ligands on leukocytes. Using recombinant soluble E-selectin constructs, we simulated the binding of E-selectin to its ligand(s) to reveal important signaling pathways triggered upon these interactions in acute myeloid leukemia (AML) cells. Since phosphorylation is the major post-translational modification, we examined the changes in the phosphorylation profile in tyrosine residues. We found a time-dependent reduction in the phosphotyrosine levels upon E-selectin binding to the AML cell line, KG-1a. The results of this study revealed two tyrosine phosphatases with altered activity after E-selectin treatment. The first is a cytoplasmic, dual-specific, phosphatase known as PTEN which is involved in controlling cell survival and proliferation. The second is CD45, which is a major component of the leukocytes cell membrane responsible for antigen receptor signaling. A more global phosphoproteomics analysis in AML cells revealed large scale changes in the phosphorylation levels after E-selectin treatment. In particular, 2259 phosphorylated proteins were identified, 530 of which portray significant changes in their phosphorylation status. The majority of those proteins are related to nuclear functions and are involved in pathways crucial for the cell cycle. Knowing that E-selectin binding stimulates chemoresistance in cancer cells, the findings of this project can contribute to the identification of multiple pathways responsible for this phenomenon and help towards the development of drugs that may inhibit such pathways in controlling disease.

No description available.

No description available.

No description available.

No description available.

No description available.

La protéine suppresseur de tumeurs PTEN (Phosphatase and tensin deleted on chromosome 10) est une phosphatase lipidique. En déphosphorylant le phosphatidylinositol (3,4,5) trisphosphate (PIP3) en PI(4,5) P2, PTEN contre-régule la voie PI3K/Akt et inhibe la prolifération. D’autres fonctions de PTEN peuvent être indépendantes de son activité phosphatase lipidique, notamment l’inhibition de la migration. Bien que PTEN soit, après p53, le suppresseur de tumeurs le plus muté dans un large panel de cancers (gliomes, prostate, sein, endomètre…), les mécanismes par lesquels ses fonctions sont régulées ne sont pas entièrement élucidés. Par une approche de double-hybride, notre équipe a identifié que les β-arrestines (β-arrs), des protéines d’échafaudage, interagissent avec PTEN. Nos travaux mettent en évidence que l’interaction entre PTEN et les β-arrs permet de moduler ses deux activités dépendantes ou non de son activité phosphatase lipidique. D’une part, les β-arrs augmentent l’activité phosphatase lipidique de PTEN in vitro. La GTPase RhoA et sa kinase d’aval ROCK activent PTEN, et ceci se fait par l’intermédiaire des β-arrs. La stimulation du récepteur à l’acide lysophosphatidique (LPA), qui active la voie RhoA/ROCK, augmente la formation du complexe PTEN/β-arrs et permet le recrutement du complexe à la membrane. Par l’effet positif sur l’activité phosphatase lipidique de PTEN, les β-arrs participent à l’inhibition d’Akt et de la prolifération dans les fibroblastes embryonnaires de souris (MEF). A l’inverse dans les gliomes U373, les β-arrs lèvent l’inhibition de la migration exercée par le domaine C2 de PTEN, indépendamment de son activité phosphatase lipidique. En aval de l’activation de RhoA induite par blessure du tapis cellulaire, les β-arrs interagissent davantage avec PTEN et rétablissent la migration des gliomes. De ce fait, les β-arrs régulent différentiellement les fonctions de PTEN importantes pour le contrôle de la prolifération cellulaire et la migration. Enfin, l’activité et la localisation de PTEN sont modulées par des interactions intramoléculaires entre ses domaines catalytiques, C2 et sa queue C-terminale régulatrice. Ces interactions régulent le passage d’une conformation fermée vers une conformation ouverte et active de PTEN. Grâce au développement d’un biosenseur de PTEN basé sur le transfert d’énergie par résonnance (RET), nous pouvons suivre pour la première fois les changements conformationnels de PTEN dans les cellules vivantes. En utilisant ce biosenseur nous montrons que la mutation des résidus impliqués dans les interactions intramoléculaires entraine des changements de conformation détectés par des variations de RET. De plus, l’activation de voies de signalisation connues pour activer PTEN, entrainent des changements conformationnels qui corrèlent avec l’augmentation de l’activité phosphatase lipidique de PTEN. Nos données montrent que le biosenseur peut être utilisé comme outil pour détecter les changements d’activité de PTEN dans les cellules vivantes. L’axe suppresseur de tumeurs/oncogène PTEN/PI3K/Akt joue un rôle essentiel dans la progression tumorale et constitue une cible thérapeutique pour le cancer. L’ensemble de nos travaux permet d’ajouter un degré de compréhension dans la régulation de PTEN, tant par les β-arrs que par l’interaction intramoléculaire et les changements conformationnels. / The Tumour Suppressor protein PTEN (Phosphatase and tensin deleted on chromosome 10) is a lipid phosphatase. By converting phosphatidylinositol (3,4,5) trisphosphate (PIP3) to PI(4,5)P2, PTEN inhibits the PI3K/Akt signalling pathway and cell proliferation. Other functions attributed to PTEN, including the inhibition of cell migration, can occur independently of its lipid phosphatase activity. Although PTEN function is dysregulated in a broad range of cancers (gliomas, prostate, breast, endometrium…), the mechanisms by which it is regulated are far from being completely elucidated. Using a two-hybrid approach, our team identified that the molecular scaffolds, β-arrestins (β-arrs), interact with PTEN.Our studies demonstrate that β-arrs modulate distinct functional outputs of PTEN that in turn are dependent or independent on its lipid phosphatase activity. β-arrs increase the lipid phosphatase activity of PTEN in vitro. The small GTPase RhoA and its downstream effector ROCK activate PTEN and this effect requires β-arrs. The stimulation of the lysophosphatidic acid receptor 1 (LPA1-R) receptor, that activates the RhoA/ROCK pathway, was found to increase the association of β-arrs with PTEN and induced plasma membrane translocation of the complex. Through their stimulatory effect on the lipid phosphatase activity of PTEN, β-arrs inhibit the PI3K/Akt pathway and proliferation of mouse embryonic fibroblasts. In contrast, in U373 glioma cells, βarrs release the brake on cell migration, which is mediated by the C2 domain of PTEN independently of its lipid phosphatase activity. Following wounding of a cell monolayer, and RhoA activation, β-arrs show increased association with PTEN, and rescue glioma cell migration. β-arrs therefore differentially regulate functions of PTEN important in the control of cell proliferation and migration.The activity and localization of PTEN are under tight control of intramolecular interactions between its regulatory C-terminal tail, and catalytic and C2 domains. These intramolecular interactions regulate a switch between a closed form of PTEN, and an open and active form that is targeted to the membrane. We have developed a resonance energy transfer (RET)-based biosensor that permits the monitoring of PTEN conformational change in live cells. Using the biosensor we demonstrate that mutation of residues implicated in the intramolecular switch produce conformational rearrangement of PTEN, detected by changes in RET. Furthermore, activation of signalling pathways known to activate PTEN, elicit conformational changes that parallel increased PTEN lipid phosphatase activity in living cells. Combined, these data demonstrate that the biosensor can be used as a tool to detect changes in PTEN tumour suppressor activity in live cells.The tumour suppressor/oncogene PTEN/PI3K/Akt axis plays a key role in tumour progression and represents a major therapeutic target in the treatment of cancer. Our studies help to further our understanding of how tumour suppressor PTEN is controlled by inter- and intramolecular interactions and provide a biosensor that can report changes in PTEN activity.