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<p>PTEN is a potent tumour suppressor protein. Aggressive and metastatic prostate cancer (PC) is associated with a reduction or loss of PTEN expression. PTEN reduction often occurs without gene mutations, and its downregulation is not fully understood. Herein, we show that PTEN is incorporated in the cargo of exosomes derived from cancer cells, and this is an exclusive characteristic of cancer cells; normal cells do not incorporate PTEN in their exosomes. We found that this process is affected by the expression of oncogenes, with activation of oncogenic molecules leading to increased PTEN incorporation into exosomes. PTEN expressed in exosomes can be transferred to other cells that have a reduction or loss of PTEN expression. The transferred PTEN is active, as cells showed a substantial increase in phosphatase activity upon treatment with PTEN-bearing exosomes. PTEN transferred through exosomes is also competent to confer tumour-suppression activity to acceptor cells. After incubation with PTEN-bearing exosomes, recipient cells exhibited decreased AKT phosphorylation, changes in the expression of cell cycle mediators indicating cell cycle arrest, and decreased proliferation. These data suggest that exosomal PTEN may be able to compensate for PTEN loss in cancer cells, by transferring the active protein to cancer cells where it can then perform its role as a tumour suppressor.</p> / Master of Science (MSc)

Primary sensory axons fail to regenerate into the spinal cord following dorsal root injury leading to permanent sensory deficits. Re-entry is prevented at the dorsal root entry zone (DREZ), the CNS-PNS interface. Current approaches for promoting DR regeneration across the DREZ have had some success, but sustained, long-distance regeneration, particularly of large-diameter myelinated axons, still remains a formidable challenge. Our lab has previously shown that induced expression of constitutively active B-RAF (kaBRAF) enhanced the regenerative competence of injured DRG neurons in adult mice. In this study, I investigated whether robust intraspinal regeneration can be achieved by selective expression of kaBRAF alone or in combination with deletion of the myelin-associated inhibitors or neuron-intrinsic growth suppressors (PTEN or SOCS3). To this end, I used LSL-kaBRAF: brn3a-CreERT2 transgenic mice in which kaBRAF can be induced selectively in sensory neurons. I have also bred LSL-kaBRAF: brn3a-CreERT2 mice with triple knock-out mice lacking Nogo, Mag and OMgp or mouse lines carrying floxed alleles of PTEN or SOCS3. Single, double, and triple conditional mice were subjected to cervical DR crush and AAV2-eGFP vectors were used to selectively label regenerating axons of large-diameter neurons. I compared the extent of regeneration at 3 weeks or 2 months after DR injury using conventional anatomical and behavioral analyses. I found that kaBRAF alone promoted axon regeneration across the DREZ but did not produce significant functional recovery by two months. Supplementary deletion of Nogo, MAG, and OMgp did not improve kaBRAF-induced regeneration. Deletion of PTEN or SOCS3 individually or in combination failed to promote axon regeneration across the DREZ. In marked contrast, simultaneous deletion of PTEN, but not SOCS3, dramatically enhanced kaBRAF-mediated regeneration enabling many more axons to penetrate the DREZ and grow deep into the spinal cord. This study shows that dual activation of BRAF-MEK-ERK and PI3K-Akt signaling is an effective strategy to stimulate robust intraspinal DR regeneration and may lead to recovery of sensory function after DR injury. / Biomedical Sciences

Chronic Hepatitis B Virus (HBV) infection is a global health problem because of its connection to acute and chronic liver diseases as well as hepatocellular carcinoma (HCC). There is increasing evidence showing that HBV contributes to HCC due to persistently high levels of trans-activating protein---hepatitis B encoded x antigen (HBxAg). Studies have shown that the HBxAg affects and alters the activity of many different transcription factors and plays an essential role in several cytoplasmic signaling transduction pathways, such as Wnt signaling pathways. One of the upregulated genes, designated URG11, was found transactivated by HBxAg. URG11 could stimulate the ß-catenin promoter and hepatocellular growth and survival which suggest that URG11 may be a regulatory element in the ß-catenin signaling pathways. microRNA148a (miR148a) was identified from two miRNA microarrays as one of the up-regulated miRNAs in cells stably expressing HBxAg or over-expressing URG11. Moreover, the expression of miR148a was also elevated in HBV-mediated HCC patient tissue samples. To study the function of miR148a, HepG2 (hepatoblastoma) and Hep3B (hepatoma) cells stably expressing HBxAg or over-expressing URG11 were transduced by recombinant lentiviruses encoding anti-miR148a. anti-miR148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Further, introduction of anti-miR148a increased PTEN protein and mRNA expression, suggesting that PTEN was suppressed by miR148a. In addition, anti-miR148a blocked the stimulation of Akt signaling, resulting in decreased expression of ß-catenin. Thus, miR148a may play a central role in HBxAg/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type. / Biology

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2005-2006 / Le neutrophile est spécialisé dans la chimiotaxie et la phagocytose. Ces deux phénomènes sont accompagnés d'une accumulation de phosphatidylinositol(3,4,5)triphosphate. Ce dernier est formé par les phosphatidylinositols 3-kinases. Leur activation et la formation de phosphatidylinositol(3,4,5)triphosphate sont bien caractérisés dans le neutrophile. La régulation du niveau de phosphatidylinositol(3,4,5)triphosphate par les lipide phosphatases est peu étudiée. Nous avons examiné le rôle des lipide phosphatases SHIP1 et PTEN suite à la stimulation du CD32a, un FcyR, dans la régulation du niveau de phosphatidylinositol(3,4,5)triphosphate. Ce dernier augmente suite à la stimulation du CD32a. La localisation cellulaire et la phosphorylation de SHIP1, mais pas de PTEN, sont modifiées en réponse à la stimulation du CD32a. Ces événements seraient dépendants des Src kinases. Par contre, ils seraient indépendants de l'activation des phosphatidylinositol 3-kinases. SHIP1, et non PTEN, pourrait donc être impliquée dans la régulation du phosphatidylinositol(3,4,5)triphosphate formé suite à la stimulation du CD32a dans le neutrophile humain.

Introdução: Apesar da identificação de fatores prognósticos clínicos e patológicos, muitos pacientes portadores de carcinoma de células renais (CCR) apresentam metástases ao diagnóstico e outros irão desenvolver recorrência local ou à distância durante o seguimento. Novos fatores prognósticos e de origem molecular têm sido avaliados no CCR, destacando-se o PTEN como um dos principais genes envolvidos na carcinogênese renal. Objetivos: Avaliar os fatores clínicos e anatomopatológicos mais significativos nas taxas de sobrevida, identificar a frequência de hipermetilação do gene PTEN através da técnica do pirosequenciamento, o impacto da hipermetilação do gene nas taxas de sobrevida global (SG) e livre de doença (SLD), como também, a associação da presença de hipermetilação com os principais fatores prognóticos. Material e métodos: Foram avaliados 137 pacientes portadores de CCR submetidos a tratamento cirúrgico do tumor primário entre 1997 e 2009. Foram considerados os dados epidemiológicos, clínicos, anatomopatológicos, de estadiamento (TNM 2004) e os obtidos da reação de pirosequenciamento. Resultados: O tempo de seguimento médio foi de 32,3 meses e mediana de 28,8 meses. Considerando o estadimento clínico, foram fatores independentes para a SG: idade (p<0,01), ASA (p=0,02), margens cirúrgicas (p=0,04), grau de Fuhrman (p=0,01), estádio clínico (p<0,001) e subtipo histológico (p<0,01). No modelo múltiplo a SLD foi influenciada únicamente pelo estádio clínico (p<0,001). Dos 137 casos analisados, hipermetilação do gene foi detectada em cinco casos (3,6%). Devido a baixa freqüência detectada optou-se por não realizar a associação da metilação do PTEN com os fatores prognósticos. Em relação às taxas de SG e SLD, de acordo com o perfil de hipermetilação do PTEN, não houve a ocorrência de nenhum evento, ou seja, morte, morte por CCR ou recorrência da doença para os cinco casos que apresentavam hipermetilação. Conclusões: A hipermetilação do xv PTEN foi detectada com baixa frequência, sugerindo a participação de outros genes ou mecanismos moleculares diferentes da metilação na inativação deste gene frequentemente envolvido na carcinogênese renal. As taxas de sobrevida não foram influenciadas pelo perfil de hipermetilação do PTEN, permanecendo o estadiamento clínico do TNM como a principal variável determinante da evolução e do risco de recidiva pelo CCR / Introduction: Despite the identification of clinical and pathological prognostic factors, many patients with renal cell carcinoma (RCC) have metastases at diagnosis and others will develop local or distant recurrence during follow-up. New prognostic factors and of molecular origin have been evaluated in RCC, highlighting PTEN, one of the main genes involved in renal carcinogenesis. Objetives: To assess the most significant clinical and pathological factors in survival rates, and identify the frequency of hypermethylation of the PTEN gene by the pyrosequencing technique, the impact of gene hypermethylation on overall survival (OS) rates and disease free interval (DFS), as well as associating presence of hypermethylation with main prognostic factors. Methods: We evaluated 137 patients with RCC that underwent surgical treatment of primary tumor between 1997 and 2009. We considered the epidemiological, clinical, pathological, staging (TNM 2004) data and those obtained from pyrosequencing. Results: Mean follow-up was of 32.3 months and the median of 28.8 months. Considering the clinical TNM stage, the OS was influenced in the multiple model by age (p < 0.01), ASA (p = 0.02), surgical margins (p = 0.04), Fuhrman´s grade (p = 0,01), clinical stage (p <0.001) and cell subtype (p < 0.01). DFS were influenced in multivariate analysis only by presence of clinical stage (p <0.001). Of the 137 cases examined, gene hypermethylation was detected in five cases (3,6%). Because of this low frequency perceived, we elected not to carry out the association of PTEN methylation with prognostic factors. Regarding OS and DFS rates, according to the hypermethylation of PTEN profile, no event occurred, that is to say death, death from RCC or disease recurrence in the five cases with hypermethylation. Conclusions: Hypermethylation of PTEN was detected with low frequency suggesting involvement of other genes or different molecular mechanisms of methylation upon inactivation of this gene, frequently involved in renal xvii carcinogenesis. Survival rates were not influenced by the hypermethylation of PTEN profile, with clinical TNM staging remaining as the main determinant for development and risk of RCC recurrence