The development of RNAi based technologies has given researchers the tools to interrogate processes as diverse as cancer biology, metabolism and organ development. Here I employ genome-wide shRNA screens to discover the genes involved in two different processes in carcinogenesis, oncogene-induced senescence [OIS] and epigenetic silencing of tumor suppressor genes [TSGs]. OIS is a poorly studied yet significant tumor suppressing mechanism in normal cells where they enter cell cycle arrest [senescence] or programmed cell death [apoptosis] in the presence of an activated oncogene. Here I employ a genomewide shRNA screen and identify a secreted protein, IGFBP7, that induces senescence and apoptosis in melanocytes upon introduction of the oncogene BRAFV600E. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 has potent pro-apoptotic and anti-tumor activity in mouse xenograft models using BRAFV600E-postive melanoma cell lines. Finally, IGFBP7 is epigenetically silenced in human melanoma samples suggesting IGFBP7 expression is a key barrier to melanoma formation. Next I investigated the factors involved in epigenetic silencing in cancer. The TSG p14ARFis inactivated in a wide range of cancers by promoter hypermethylation through unknown mechanisms. To discover p14ARF epigenetic silencing factors, I performed a genome-wide shRNA screen and identified ZNF304, a zinc finger transcription factor that contains a Krüppel-associated box [KRAB] repressor domain. I show that ZNF304 binds to the p14ARF promoter and recruits a KRAB co-repressor complex containing KAP1, SETDB1 and DNMT1 for silencing. We find oncogenic RAS signaling to promote the silencing of p14ARF by USP28-mediated stabilization of ZNF304. In addition I find ZNF304 to be overexpressed in human colorectal cancers and responsible for hypermethylation of over 50 TSGs known as Group 2 CIMP marker genes. My findings establish ZNF304 as a novel oncogene that directs epigenetic silencing and facilitates tumorigenicity in colorectal cancer.

No description available.

INTRODUÇÃO: O Transtorno Bipolar (TB) é um transtorno psiquiátrico crônico e debilitante e sua etiologia e patologia ainda não são completamente conhecidos, apesar de um componente genético importante ser evidenciado em estudos de família, adoção e gêmeos. Recentemente, o TB tem sido relacionado a um processo de envelhecimento acelerado, com alguns estudos mostrando telômeros encurtados nesta população. O objetivo do presente estudo foi investigar a associação entre o comprimento telomérico, um dos parâmetros do processo de envelhecimento celular, com a ausência ou presença de TB em famílias com muitos membros afetados, além de associar a sintomatologia clínica e outras variáveis a esse parâmetro. Procurou-se também avaliar as influências genéticas e ambientais sobre o comprimento telomérico nessas famílias, estimando-se a herdabilidade desta característica. MÉTODOS: O comprimento telomérico (T) foi mensurado em uma amostra de 143 indivíduos de 22 famílias (60 deles com TB), em relação a um gene de cópia única (S) - beta-globina, através do método de PCR (Polymerase Chain Reaction) em tempo real quantitativo, no qual forneceu uma proporção do número de cópias de T por S (razão T/S). Considerando a estrutura familiar na análise estatística foi ajustado para cada análise o modelo misto poligênico. RESULTADOS: O efeito do TB no comprimento dos telômeros foi pequeno, não tendo sido observada uma associação estatisticamente significante entre TB e comprimento telomérico quando comparado com familiares saudáveis (p > 0,05). No entanto, observou-se associação do comprimento telomérico à covariável ideação suicida (p = 0,02) e à interação entre ideação suicida e curso da doença (p = 0,02). Associação do comprimento telomérico com idade materna e TB também foi observada (p < 0,05). Por fim, estimou-se em 68% a herdabilidade do comprimento telomérico nas 22 famílias do estudo. CONCLUSÕES: A teoria do envelhecimento acelerado em TB, vista pela óptica do comprimento dos telômeros, não pôde ser confirmada no presente estudo, pois não foi encontrada diferença no comprimento telomérico entre indivíduos saudáveis e com TB nas famílias. Por outro lado, covariáveis que indicam gravidade da doença, como a ideação suicida e a interação entre ideação suicida e curso da doença foram associadas ao comprimento telomérico (p < 0,05), ou seja, um encurtamento telomérico foi correlacionado à gravidade clínica do TB. Associação do comprimento telomérico com idade materna e TB (p < 0,05) sugeriu que a idade materna avançada não só pode ser um marcador de longevidade, como também o fenótipo TB pareceu reforçar essa condição. Por fim, a alta herdabilidade estimada do comprimento telomérico (0,68) revelou uma importante variabilidade genética desse fenótipo entre as famílias do estudo. Em súmula, este é o primeiro estudo que relatou uma associação entre ideação suicida, curso da doença, idade materna e comprimento telomérico em famílias com vários membros afetados pelo TB. Outras investigações independentes são necessárias para confirmar esses resultados preliminares / BACKGROUND: Bipolar Disorder (BD) is a debilitating and chronic mental illness. It is etiology and pathology are not completely known yet, despite the evidence of an important genetic component from family, twin and adoption studies. Recently, BD has been related to a process of accelerated aging, with some studies showing shortened leukocyte telomeres in this population. The purpose of the present study was to investigate the association between leucocyte telomere length (LTL) in BD patients compared with healthy relatives of 22 families with several affected members by this illness, besides associating clinical symptomatology and other covariates with this parameter. It was also examined the genetic and environmental influences on telomere length trait in these BD families, using a variance component approach, by estimating the heritability of this trait as well as covariate effects. METHODS: Telomere length (T) was estimated in a sample of 143 individuals, including 60 BD patients from 22 families, which was measured in relation to the single copy gene (S) - beta-globin gene, using a singleplex real time PCR (Polymerase Chain Reaction), providing a ratio of number of copies of T by S (T/S ratio). Taking in consideration the family structure, the statistical analysis was adjusted for the polygenic mixed model. RESULTS: The effect of BD illness in telomere length was small and we found no association between BD group and LTL (p > 0.05). However, LTL was associated with the variable suicidal ideation (p = 0.02) and interaction between suicidal ideation and course of disorder (p = 0.02). Association of LTL and maternal age and BD was also observed (p < 0.05). In addition, an important genetic component for telomere length was also observed (heritability = 0.68) in these families. CONCLUSIONS: The hypothesis of accelerated aging in BD, investigating the telomere length as one of its components, was not confirmed in our study. We found no difference between LTL and BD in our family group. However, using covariates that indicate severity of disease, both suicidal ideation and interaction between suicidal ideation and course of disorder were statistically significant with LTL, showing that shorter LTL was associated with worse clinical course (p < 0.05) and suicidal ideation (p < 0.05) in BD patients. Association of LTL with maternal age and BD (p < 0.05) suggests that advanced maternal age may not only be a marker of longevity, but also the BD phenotype may reinforce this condition. A high heritability for telomere length (0.68) also suggests an important genetic variability of this trait presented among those families. To our knowledge, this is the first study that found association between suicidal ideation, course of disorder, maternal age and LTL in families with several members affected by BD. Further investigations, including replication studies in other BD families, are needed to confirm these new findings

HIV associado a desordens neurocognitivas (HAND) continua a ser um grave problema atualmente devido à alta prevalência de suas formas mais brandas. Indivíduos HIV+ possuem o comprimento dos telômeros significativamente mais curtos nas células mononucleares do sangue periférico e células T CD8+, quando comparados aos indivíduos HIV negativos. Diante do exposto, o objetivo deste estudo foi avaliar a associação do comprimento dos telômeros de leucócitos em indivíduos infectados pelo HIV com deficiências cognitivas, pois ainda é um assunto bastante controverso. Métodos: Um total de 73 pacientes infectados pelo HIV-1 de ambos os sexos, com idades entre 20 a 60 anos, participaram deste estudo. Entre 19 indivíduos HIV(+) sem comprometimento cognitivo e 54 indivíduos HIV(+) com distúrbios neurocognitivos: 29 alteração neurocognitiva assintomático (ANI), 15 comprometimento neurocognitivo leve a moderado (MND) e, 10 demência associada ao HIV (HAD); 118 indivíduos HIV negativos formaram o grupo controle. Todos os participantes foram submetidos a uma série de testes neuropsicológicos previamente validados. Determinou-se a carga viral de HIV-1 nas células do líquido cefalorraquidiano (LCR) e em PBMC. Utilizou-se DNA a partir de leucócitos periféricos para calcular o comprimento de telômeros por PCR em tempo real. Resultados: O comprimento dos telômeros não foi associado com gêneros e diminuiu com a idade, independentemente do status de HIV. Indivíduos infectados pelo HIV-1com formas mais leves de deficiência neurocognitiva apresentaram um comprimento dos telômeros reduzida em comparação com pacientes HIV+ sem comprometimento neurocognitivo. Não houve correlação entre a carga viral plasmática e o tamanho dos telômeros. Conclusões: Nossos resultados sugerem que o comprimento dos telômeros pode ser considerado um marcador de senescência celular em indivíduos com alterações neurocognitivas / HIV associated neurocognitive disorders (HAND) remains a serious problem today because of the high prevalence of its milder forms. HIV + individuals have the length substantially shorter telomeres in peripheral blood mononuclear cells and CD8 + T cells compared to HIV negative individuals. Given the above, the objective of this study was to evaluate the association of telomere length of leukocyte (LTL) in HIV-infected individuals with cognitive disabilities because it is still a very controversial subject. Methods: A total of 73 patients infected with HIV-1 of both sexes, aged 20 to 60 years participated in this study. Among 19 HIV patients (+) without cognitive impairment and 54 HIV patients (+) with neurocognitive disorders: 29 asymptomatic neurocognitive disorder (ANI), 15 mild neurocognitive disorder to moderate (MND) and 10 HIVassociated dementia (HAD); 118 HIV-negative individuals formed the control group. All participants underwent a series of previously validated neuropsychological tests. Determined if the viral load of HIV-1 in cerebrospinal fluid cells (CSF) and in PBMC. We used DNA from peripheral leukocytes to calculate the length of telomeres by real time PCR. Results: The telomere length was not associated with genres and decreased with age, irrespective of HIV status. HIV-1-infected individuals with milder forms of neurocognitive impairment had a significantly length of telomeres reduced compared to HIV + patients without neurocognitive impairment. There was no correlation between plasma viral load and the size of telomeres. Conclusions: Our results suggest that telomere length can be considered a marker of cellular senescence in individuals with neurocognitive abnormalities

HIV associado a desordens neurocognitivas (HAND) continua a ser um grave problema atualmente devido à alta prevalência de suas formas mais brandas. Indivíduos HIV+ possuem o comprimento dos telômeros significativamente mais curtos nas células mononucleares do sangue periférico e células T CD8+, quando comparados aos indivíduos HIV negativos. Diante do exposto, o objetivo deste estudo foi avaliar a associação do comprimento dos telômeros de leucócitos em indivíduos infectados pelo HIV com deficiências cognitivas, pois ainda é um assunto bastante controverso. Métodos: Um total de 73 pacientes infectados pelo HIV-1 de ambos os sexos, com idades entre 20 a 60 anos, participaram deste estudo. Entre 19 indivíduos HIV(+) sem comprometimento cognitivo e 54 indivíduos HIV(+) com distúrbios neurocognitivos: 29 alteração neurocognitiva assintomático (ANI), 15 comprometimento neurocognitivo leve a moderado (MND) e, 10 demência associada ao HIV (HAD); 118 indivíduos HIV negativos formaram o grupo controle. Todos os participantes foram submetidos a uma série de testes neuropsicológicos previamente validados. Determinou-se a carga viral de HIV-1 nas células do líquido cefalorraquidiano (LCR) e em PBMC. Utilizou-se DNA a partir de leucócitos periféricos para calcular o comprimento de telômeros por PCR em tempo real. Resultados: O comprimento dos telômeros não foi associado com gêneros e diminuiu com a idade, independentemente do status de HIV. Indivíduos infectados pelo HIV-1com formas mais leves de deficiência neurocognitiva apresentaram um comprimento dos telômeros reduzida em comparação com pacientes HIV+ sem comprometimento neurocognitivo. Não houve correlação entre a carga viral plasmática e o tamanho dos telômeros. Conclusões: Nossos resultados sugerem que o comprimento dos telômeros pode ser considerado um marcador de senescência celular em indivíduos com alterações neurocognitivas / HIV associated neurocognitive disorders (HAND) remains a serious problem today because of the high prevalence of its milder forms. HIV + individuals have the length substantially shorter telomeres in peripheral blood mononuclear cells and CD8 + T cells compared to HIV negative individuals. Given the above, the objective of this study was to evaluate the association of telomere length of leukocyte (LTL) in HIV-infected individuals with cognitive disabilities because it is still a very controversial subject. Methods: A total of 73 patients infected with HIV-1 of both sexes, aged 20 to 60 years participated in this study. Among 19 HIV patients (+) without cognitive impairment and 54 HIV patients (+) with neurocognitive disorders: 29 asymptomatic neurocognitive disorder (ANI), 15 mild neurocognitive disorder to moderate (MND) and 10 HIVassociated dementia (HAD); 118 HIV-negative individuals formed the control group. All participants underwent a series of previously validated neuropsychological tests. Determined if the viral load of HIV-1 in cerebrospinal fluid cells (CSF) and in PBMC. We used DNA from peripheral leukocytes to calculate the length of telomeres by real time PCR. Results: The telomere length was not associated with genres and decreased with age, irrespective of HIV status. HIV-1-infected individuals with milder forms of neurocognitive impairment had a significantly length of telomeres reduced compared to HIV + patients without neurocognitive impairment. There was no correlation between plasma viral load and the size of telomeres. Conclusions: Our results suggest that telomere length can be considered a marker of cellular senescence in individuals with neurocognitive abnormalities

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed on June 26, 2009). Includes bibliographical references.

INTRODUÇÃO: O Transtorno Bipolar (TB) é um transtorno psiquiátrico crônico e debilitante e sua etiologia e patologia ainda não são completamente conhecidos, apesar de um componente genético importante ser evidenciado em estudos de família, adoção e gêmeos. Recentemente, o TB tem sido relacionado a um processo de envelhecimento acelerado, com alguns estudos mostrando telômeros encurtados nesta população. O objetivo do presente estudo foi investigar a associação entre o comprimento telomérico, um dos parâmetros do processo de envelhecimento celular, com a ausência ou presença de TB em famílias com muitos membros afetados, além de associar a sintomatologia clínica e outras variáveis a esse parâmetro. Procurou-se também avaliar as influências genéticas e ambientais sobre o comprimento telomérico nessas famílias, estimando-se a herdabilidade desta característica. MÉTODOS: O comprimento telomérico (T) foi mensurado em uma amostra de 143 indivíduos de 22 famílias (60 deles com TB), em relação a um gene de cópia única (S) - beta-globina, através do método de PCR (Polymerase Chain Reaction) em tempo real quantitativo, no qual forneceu uma proporção do número de cópias de T por S (razão T/S). Considerando a estrutura familiar na análise estatística foi ajustado para cada análise o modelo misto poligênico. RESULTADOS: O efeito do TB no comprimento dos telômeros foi pequeno, não tendo sido observada uma associação estatisticamente significante entre TB e comprimento telomérico quando comparado com familiares saudáveis (p > 0,05). No entanto, observou-se associação do comprimento telomérico à covariável ideação suicida (p = 0,02) e à interação entre ideação suicida e curso da doença (p = 0,02). Associação do comprimento telomérico com idade materna e TB também foi observada (p < 0,05). Por fim, estimou-se em 68% a herdabilidade do comprimento telomérico nas 22 famílias do estudo. CONCLUSÕES: A teoria do envelhecimento acelerado em TB, vista pela óptica do comprimento dos telômeros, não pôde ser confirmada no presente estudo, pois não foi encontrada diferença no comprimento telomérico entre indivíduos saudáveis e com TB nas famílias. Por outro lado, covariáveis que indicam gravidade da doença, como a ideação suicida e a interação entre ideação suicida e curso da doença foram associadas ao comprimento telomérico (p < 0,05), ou seja, um encurtamento telomérico foi correlacionado à gravidade clínica do TB. Associação do comprimento telomérico com idade materna e TB (p < 0,05) sugeriu que a idade materna avançada não só pode ser um marcador de longevidade, como também o fenótipo TB pareceu reforçar essa condição. Por fim, a alta herdabilidade estimada do comprimento telomérico (0,68) revelou uma importante variabilidade genética desse fenótipo entre as famílias do estudo. Em súmula, este é o primeiro estudo que relatou uma associação entre ideação suicida, curso da doença, idade materna e comprimento telomérico em famílias com vários membros afetados pelo TB. Outras investigações independentes são necessárias para confirmar esses resultados preliminares / BACKGROUND: Bipolar Disorder (BD) is a debilitating and chronic mental illness. It is etiology and pathology are not completely known yet, despite the evidence of an important genetic component from family, twin and adoption studies. Recently, BD has been related to a process of accelerated aging, with some studies showing shortened leukocyte telomeres in this population. The purpose of the present study was to investigate the association between leucocyte telomere length (LTL) in BD patients compared with healthy relatives of 22 families with several affected members by this illness, besides associating clinical symptomatology and other covariates with this parameter. It was also examined the genetic and environmental influences on telomere length trait in these BD families, using a variance component approach, by estimating the heritability of this trait as well as covariate effects. METHODS: Telomere length (T) was estimated in a sample of 143 individuals, including 60 BD patients from 22 families, which was measured in relation to the single copy gene (S) - beta-globin gene, using a singleplex real time PCR (Polymerase Chain Reaction), providing a ratio of number of copies of T by S (T/S ratio). Taking in consideration the family structure, the statistical analysis was adjusted for the polygenic mixed model. RESULTS: The effect of BD illness in telomere length was small and we found no association between BD group and LTL (p > 0.05). However, LTL was associated with the variable suicidal ideation (p = 0.02) and interaction between suicidal ideation and course of disorder (p = 0.02). Association of LTL and maternal age and BD was also observed (p < 0.05). In addition, an important genetic component for telomere length was also observed (heritability = 0.68) in these families. CONCLUSIONS: The hypothesis of accelerated aging in BD, investigating the telomere length as one of its components, was not confirmed in our study. We found no difference between LTL and BD in our family group. However, using covariates that indicate severity of disease, both suicidal ideation and interaction between suicidal ideation and course of disorder were statistically significant with LTL, showing that shorter LTL was associated with worse clinical course (p < 0.05) and suicidal ideation (p < 0.05) in BD patients. Association of LTL with maternal age and BD (p < 0.05) suggests that advanced maternal age may not only be a marker of longevity, but also the BD phenotype may reinforce this condition. A high heritability for telomere length (0.68) also suggests an important genetic variability of this trait presented among those families. To our knowledge, this is the first study that found association between suicidal ideation, course of disorder, maternal age and LTL in families with several members affected by BD. Further investigations, including replication studies in other BD families, are needed to confirm these new findings

Glioma therapy relies on induction of cytotoxicity; however, the current combination of surgery, irradiation (IR) and temozolomide (TMZ) treatment does not result in a long-term cure. Our lab previously demonstrated that a small population of glioma cells enters a transient cell cycle arrest in response to chemotherapy. Treatment with TMZ significantly decreases initial neurosphere formation; however, after a short recovery period, a small number of cells resume neurosphere formation and repopulate the culture. This recovery of neurosphere growth recapitulates the inevitable glioma recurrence in the clinic. The focus of our laboratory is to study direct-target therapies that can be combined with TMZ to inhibit neurosphere recovery. The Notch pathway is a promising target because it is involved in cell growth and survival. Here, we demonstrate that blocking the Notch pathway using gamma-secretase inhibitors (GSIs) enhances TMZ treatment. The combination of TMZ and GSI treatments targets the cells capable of recovery. TMZ + GSI treated cells do not recover and are no longer capable of self-renewal. Interestingly, recovery is inhibited when the GSI is administered 24 hrs after TMZ treatment, demonstrating a sequence-dependent mechanism. TMZ + GSI treatment also decreases tumorigenicity. When glioma cell lines were treated in vitro and implanted in NU/NU nude mice, TMZ + GSI treatment extended latency and greatly increased survival. In addition, in vivo TMZ + GSI treatment completely blocked tumor progression and resulted in the loss of a palpable tumor in 50% of mice, while none of the TMZ-only treated mice survived. TMZ + GSI treated cultures and xenografts display a senescent phenotype. Cultures treated with TMZ + GSI have decreased proliferation, but no increase in cell death. We observed an increase in the number of cells expressing senescence-associated β-galactosidase in vitro and in vivo. This demonstrates that inhibition of the Notch pathway shifts TMZ-treated cells from a transient cell cycle arrest into a permanent senescent state. Senescent cells can stimulate the innate immune system. Here we demonstrate that TMZ + GSI treatment increases phagocytosis in vitro. New therapy combinations, such as TMZ + GSI, are arising in the field of therapy-induced senescence (TIS). Overall, this data demonstrates the importance of the Notch pathway in chemoprotection and maintenance of TMZ-treated gliomas. The addition of GSIs to current treatments is a promising target-directed therapy to decrease the rate of brain tumor recurrence by inducing senescence and tumor clearance.

Cellular senescence is thought to play an essential role in many biological functions including tumor suppression and organismal aging. Senescent cells, which are permanently removed from the cell cycle, can be found both in vivo in many different tissue types and in vitro within cultures of non-immortalized cells. Despite their inability to proliferate, these cells persist and remain metabolically active for indefinite periods of time. This physiologic process occurs in response to a variety of cellular insults including oxidative stress, shortened telomeres, constitutive oncogene expression, and DNA damage, and can be initiated by upregulation of one of the two known senescent pathways, involving p16/Rb or p53/p21. The senescent cell phenotype is also characterized by changes to cell and nuclear morphology and to the secretory profile of the cell. Related to changes in nuclear morphology, epigenetic modifications to the packaging of DNA are thought to be key to the initiation and maintenance of the senescence program. While a large number of earlier studies focused on the findings that senescent cells gain regions of condensed heterochromatin, often in the form of Senescent Associated Heterochromatin Foci (SAHF), this thesis work shows that there is a marked loss of heterochromatin in the peri/centromeric regions of the genome. In fact, both α-satellite and satellite II sequences across the genome distend in a striking and unanticipated fashion; this can be readily visualized by fluorescence in situ hybridization (FISH) as their structure changes from a condensed spot to highly elongated and fine thread-like signals. We have termed this exceptional decondensation of constitutive heterochromatin Senescence Associated Distension of Satellites (SADS). Importantly, a series of experiments shows that SADS is both a consistent and an early event in the cell senescence process, which occurs as a result of every senescence induction method examined. We also observed that this distension was characteristic of both human and murine cells and in vivo in human benign Prostatic Intraepithelial Neoplasia (PIN) tissue. Furthermore, unlike SAHF formation, SADS can occur due to the activation of either of the two senescence pathways, p16/Rb or p53/p21. Additionally, the cytological dimensions of the thread-like satellite signals indicates that SADS represents “unraveling” of DNA on an unprecedented scale. Thus, it was surprising that this event was not facilitated by changes to several canonical histone modifications associated with condensed heterochromatin, namely H3K9Me3, H3K27Me3, or H3K4Me3, nor is it caused by loss of DNA methylation. Consequently, we believe that this marked distension of satellite DNA is due to changes in higher-order folding of the chromatin fiber. This is important for understanding fundamental events in the cell senescence process, but also provides a unique system for study of chromatin packaging that may provide new insights into the organization of DNA well beyond nucleosome packaging and the ten nanometer fiber. In fact, initial super resolution images of SADS suggest that the satellite sequences may be organized into domains or “globules”. Hence, we suggest that the changes to satellite sequence packaging may be facilitated by changes to higher-order nuclear structural proteins, such as LaminB1, which is reduced in senescent cells. Finally, this work provides analysis of the literature and preliminary experiments to consider the possibility that there are increased levels of cell senescence in Down syndrome (trisomy 21) cells. As individuals with Down syndrome (DS) experience many manifestations of premature aging (including early-onset Alzheimer’s Disease), have a resistance to solid tumor formation, are more susceptible to oxidative stress, and are trisomic for several genes implicated in causing senescence, our analysis provides plausibility for the hypothesis that accelerated rates of senescence may play a significant role in DS physiology. We also provide results of preliminary studies and outline the next steps for experimentation, using DS fibroblasts and a unique genetically engineered DS iPS cell system. As a final note, the quantification of cell senescence in trisomic versus disomic cells for these experiments relies substantially on the new single-cell marker of senescence discovered and established by this theses work, the Senescence-Associated Distension of Satellites.

In response to telomere shortening, oxidative stress, DNA damage or aberrant activation of oncogenes, normal somatic cells exit the cell cycle and enter an irreversible growth arrest termed senescence. The limited proliferative capacity imposed by senescence on cells impedes the accumulation of mutations necessary for tumorigenesis and prevents proliferation of cells at risk of neoplastic transformation. Opposite to the tumor suppressor function, accumulation of senescent cells in adult organisms is thought to contribute to aging by depleting the renewal capacity of tissues and stem/progenitor cells, and by interfering with tissue homeostasis and functions. The Antagonistic Pleiotropy Theory of senescence proposes that senescence is beneficial early in life by acting as a tumor suppressor, but harmful late in life by contributing to aging. Recent studies have provided evidence strongly supporting the tumor suppressor function of senescence, however, direct evidence supporting the role of senescence in aging remains largely elusive. In this thesis, I describe studies to test the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging. The approach that I have taken is to alter the senescence response in vivo by changing the expression of a senescence regulator in mice. The consequence of altered senescence response on tumorigenesis and stem cell self-renewal was investigated. The senescence regulator I studied is Smurf2, which has been shown previously to activate senescence in culture. I hypothesized that the senescence response will be impaired by Smurf2 deficiency in vivo. Consequently, Smurf2-deficient mice will develop tumors at an increased frequency, but also gain enhanced self-renewal capacity of stem/progenitor cells with age. I generated a Smurf2-deficient mouse model, and found that Smurf2 deficiency attenuated p16 expression and impaired the senescence response in primary cells and tissues. Smurf2-deficient mice exhibited an increased susceptibility to spontaneous tumorigenesis, indicating that Smurf2 is a tumor suppressor. At the premalignant stage of tumorigenesis, a defective senescence response was documented in the Smurf2-deficient mice, providing a mechanistic link between impaired senescence response and increased tumorigenesis. The majority of tumors developed in Smurf2-deficent mice were B-cell lymphomas with an origin in germinal centers of the spleen and a phenotype resembling human diffuse large B-cell lymphoma (DLBCL). I discovered that Smurf2 mediated ubiquitination of YY1, a master regulator of germinal centers. Stabilization of YY1 in the absence of Smurf2 was responsible for increased cell proliferation and drove lymphomagenesis in Smurf2-deficient mice. Consistently, a significant decrease of Smurf2 expression was observed in human primary DLBCL samples, and more importantly, a low level of Smurf2 expression in DLBCL correlated with poor survival prognosis. Moreover, I found that hematopoietic stem cells (HSCs) in Smurf2-deficient mice had enhanced function compared to wild-type controls. This enhanced stem cell function was associated with increased cell proliferation and decreased p16 expression, suggesting that defective senescence response in Smurf2-deficient mice leads to increased self-renewal capacity of HSCs. My study, for the first time, offers direct genetic evidence of an important tumor suppressor function for Smurf2 as well as its function in contributing to stem cell aging. Collectively, these findings provide strong evidence supporting the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging.