The family of proteins known as heterogeneous nuclear ribonucleoproteins (hnRNPs) is large and diverse. Often, one and the same hnRNP will perform multiple cellular functions, leading to their description as “multifunctional proteins”. The two hnRNPs known as hnRNP A1 and hnRNP C1/C2 are multifunctional proteins found to affect the transcription, splicing, stability, and translation of specific genes’ mRNA. They are implicated in carcinogenesis, apoptosis, and DNA damage response mechanisms. The aims of this thesis were to study the hnRNP A1 and hnRNP C1/C2 dependent regulation of two highly stress responsive genes, the tumor suppressor p53 and the cytochrome P450 enzyme Cyp2a5/CYP2A6. We identified hnRNP C1/C2 as a DNA damage induced binding protein towards the coding region of p53 mRNA, and found that while a specific cis binding site appears to have a positive function in p53 expression, interaction of hnRNP C1/C2 with this site represses the expression. The data suggest that two distinct molecular mechanisms exist for the down-regulation of p53 by hnRNP C1/C2. One mechanism, active during transcriptional stress, is dependent upon the aforementioned site, and the other, independent. We discuss how hnRNP C1/C2 dependent repression of p53 may play a role in apoptosis. The data presented here further suggest that the transcriptional and post-transcriptional processes controlling the expression of the murine Cyp2a5 gene are linked via hnRNP A1, by performing functions in the nucleus as a transcription factor, or in the cytoplasmic compartment as a trans factor bound to the 3’UTR of the mRNA as needed. Our studies of the human ortholog of this gene, CYP2A6, suggest that this gene is regulated post-transcriptionally in a manner similar to that of its murine counterpart, via changes in mRNA stability and interaction of hnRNP A1 with its 3’ UTR.

The von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is transmitted in an autosomal dominant manner. The disease is characterized by the formation of highly angiogenic tumors in many organs but the main causes of mortality are renal cell carcinomas and hemangioblastomas. Mutations in the VHL protein are responsible for the pathogenesis of the disease. VHL associates with elongin Band C to form the VBC complex. The cullin 2 protein (CUL2) and ring box protein 1 (RBX1) also associate with the VBC complex to form an E3 ubiquitin ligase involved in the ubiquitination and subsequent degradation of the hypoxia inducible transcription factor (HIF2alpha). Mutations in VHL that abrogate its E3 ligase activity lead to increased levels ofHIF2alpha and the subsequent accumulation of pro-proliferative and pro-angiogenic HIF2alpha target genes. VHL also has an important function in the regulation of extracellular matrix (ECM) assembly which is independent of its HIF2alpha regulation pathway. VHL's regulation of ECM assembly was shown to have important consequences for tumor angiogenesis and cell invasion. It was shown to be necessary for the proper assembly of a fibronectin matrix and was most recently found to interact with collagen IV alpha 2 (COL4A2). The aim of this thesis is to further characterize the VHL-COL4A2 interaction. VHL was shown to interact directly and specifically to COL4A2 and is necessary for proper COL4A2 matrix assembly. The association of VHL with COL4A2 appears to be independent of its functions as an E3 ubiquitn ligase and CUL2 was identified as part of the VBC complex that associates with collagen IV (COL4). Furthermore, a strategy to identify the binding site of VHL on COL4A2 has been employed and is in progress. These experiments represent the beginning of investigations into the novel interaction between VHL and COL4A2.

The HPV E6-p53 interaction is well-understood, but not for all high-risk HPV types. In addition, HPV E6 p53-independent functions are gaining recognition for their importance in cellular transformation but require clarification. Thus, the aim of this study was two-fold: (1) to gain insight into the p53-E6 interaction for high-risk HPV-33 and, (2) to explore how high-risk HPV E6 proteins targets cellular MAGI-3 for degradation. / In vivo and in vitro results indicated that E6 from HPV types 18 and 33 interacted similarly with p53 although, variants of the HPV-33 E6 prototype demonstrated interesting disparities. Of note was HPV-33 E6 variant 2, which degraded p53 more efficiently than prototype HPV-33 E6 and HPV-18 E6. The E6 protein from HPV types 18 and 33 also potently degraded MAGI-3 via a different pathway than that used for p53. Specifically, proteasome inhibition did not interfere with MAGI-3 degradation and MAGI-3 was not ubiquitinated in the presence of the E6 protein. / Therefore, the results described herein enhance our understanding of high-risk HPV type 33 E6 and the E6-MAGI-3 interaction.

The p53 tumor suppressor protein is upregulated in response to DNA damage and other stress signals. The upregulation of p53 involves freeing it from negative regulation imposed by Mdm2 and MdmX (Mdm4). Accumulating evidence indicates that phosphorylation of Mdm proteins by different stress-activated kinases such as ATM or c-Abl significantly impacts p53 functions. We have previously shown that ATM phosphorylation of Mdm2 Ser394 is required for robust p53 stabilization and activation following DNA damage. This dissertation describes in vivo examination of the mechanism by which Mdm2 Ser394 phosphorylation impacts p53 activities and its contribution to suppression of oncogene and DNA damage-induced tumors. We determine that phosphorylation of Mdm2 Ser394 regulates p53 activity by modulating Mdm2 stability and paradoxically delays Myc-driven lymphomagenesis while increasing lymphomagenesis in sub-lethally irradiated mice. c-Abl phosphorylates the residue neighboring Mdm2 Ser394, Mdm2 Tyr393. This dissertation describes the generation of a novel Mdm2Y393F mutant mouse to determine if c-Abl phosphorylation of Mdm2 regulates p53-mediated DNA damage responses or tumor suppression in vivo. Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes. The studies presented herein further our understanding of the mechanisms by which DNA damage-associated kinases stabilize and activate p53, and influence p53-dependent responses and tumor suppression. A better understanding of the in vivo effects of Mdm2 phosphorylation may facilitate the development of novel therapeutics capable of stimulating p53 anti-tumor activity or alleviating p53- dependent toxicities in non-malignant tissues.

For 30 years, the tumor suppressor p53 has been a subject of intense research in nearly every discipline of scientific inquiry. While numerous surprising roles for p53 in health and disease are uncovered each year, the central role of its activation in preventing neoplastic transformation has been and will remain at the forefront of p53 research as investigators work to address an unexpectedly complex question—precisely how does p53 integrate upstream stress signals to coordinate activation of its target genes in response to stress? One manner in which to address this question is at the level of transcription initiation—after upstream signals converge on p53 and produce a number of pools of post-transcriptionally modified p53, how exactly are specific target promoters activated in such a sensitive, context-specific manner? The work presented herein aims to address the role of histone acetylation at the p21 promoter—a critical mediator of G1/S arrest—by the P/CAF acetyltransferase in response to a variety of p53-activating stresses. We show that depletion of P/CAF strongly inhibits p21 expression in response to a variety of stresses, despite normal stabilization of p53 and recruitment to target promoters. This defect in p21 expression correlates closely with abrogation of stress-induced cell-cycle arrest. Strikingly, a p53 allele lacking putative P/CAF acetylation sites was still able to direct p21 expression, which was still dependent upon P/CAF. We show further that histone acetylation at H3K14 at the p21 promoter following stress is dependent upon P/CAF. Rescue of p21 expression with wild-type P/CAF or a ∆HAT point mutant indicates that P/CAF requires an intact HAT domain, suggesting that histone acetylation at H3K14 is catalyzed by P/CAF HAT activity, not the molecular bridging of a heterologous HAT by P/CAF. Furthermore, RNA polymerase II (RNAP II) was present at the p21 proximal promoter under all basal and stress conditions, but elongation of RNAP II after stress required the presence of P/CAF. These data indicate that H3K14 acetylation by P/CAF closely correlates with the activation status of the p21 promoter, and may be necessary for activation of a larger subset of p53-responsive promoters. In addition to its critical role in p21 expression, we noted that p53 stabilization and cell-cycle arrest in response to p14ARF, but not other p53-stabilizing stresses, were also dependent on P/CAF. Cell-cycle arrest induced by p16INK4A was intact after P/CAF ablation, indicating a role for P/CAF in cell-cycle arrest specific to p14ARF-p53 signaling. Basal MDM2 levels were unaffected by P/CAF knockdown, as were p53- MDM2 and ARF-MDM2 complexes. A preliminary analysis of MDM2 localization was inconclusive, due to vastly different quantities of MDM2 in different conditions making analysis of subcellular localization difficult; however, the role of P/CAF in the relocalization of MDM2 to the nucleolus by p14ARF could potentially explain the defect in p53 stabilization, and should be explored further. These observations, underscored by recent reports that P/CAF undergoes loss of heterozygosity in several tumor types, suggest that P/CAF plays a critical role in p53-mediated cell-cycle arrest through multiple, independent mechanisms. Further study should clarify whether P/CAF is lost in tumors maintaining wild-type p53, and whether its reintroduction into these tumors confers any potential therapeutic benefit.

No description available.

No description available.

O adenocarcinoma pancreático é a neoplasia maligna mais comum do pâncreas. A alta taxa de mortalidade deve-se ao diagnóstico tardio e a alta agressividade do tumor. Freqüentemente observam-se indivíduos com neoplasias de mesmo estadio apresentarem sobrevivência diferente. Isso demonstra a necessidade de incluir mais variáveis na caracterização da doença. O processo de angiogênese é essencial para o crescimento tanto do tumor primário, quanto para o metastático. A medida da densidade intratumoral da microvasculatura (DMV) por imunoistoquímica é o método mais confiável para medir a atividade angiogênica tumoral. A perda da função do gene p53 influencia a resposta à quimio e à radioterapia além de regular a angiogênese. A sobrevivência está inversamente relacionada à positividade do p53 e à DMV em neoplasias de mama, pulmão, ovários, estômago, cólon, laringe e bexiga. No adenocarcinoma pancreático os resultados são controversos. Idealizou-se essa pesquisa retrospectiva analisando-se dados clínicos e os resultados de estudos imunoistoquímicos obtidos de adenocarcinomas de pâncreas ressecados com intenção curativa. Analisou-se dados clínicos, patológicos, re-estadiamento e resultados da DMV e da expressão do gene p53 em 49 pacientes. A densidade média de microvasos foi de 46,2 vasos/mm2 sendo que esse valor foi utilizado para dividir os pacientes em grupos de baixa ou alta densidade de vasos. A coloração para p53 nuclear foi considerada positiva em 20 de 49 pacientes (40,8%). A DMV foi significativamente maior nos pacientes com tumores maiores que 3,0 cm e nos pacientes com ressecções incompletas. A expressão do gene p53 e a DMV, não foram fatores preditivos da sobrevivência pós-operatória. Não foi possível verificar relação entre a expressão do gene p53 e a densidade da microvasculatura tumoral / The prognostic significance of microvessel density and the p53 expression was evaluated. Between 1993 and 2006, 49 patients with pancreatic adenocarcinoma were ressected with curative intention. Specimens were stained immunohistochemically with antibodies anti- p53 anti-CD34. Microvessel density (MVD) was assessed scanning ten areas of the tumoral section and counted at a high power in an adequate area. The MVD ranged from 21,2 to 54,2 vessels/mm2 (mean 46,2 vessels/mm2). Specific nuclear staining for p53 was determined positive in 20 patients (40,8%). The overall median survival was 24,1 months after resection and there was no difference in survival rates according to the MVD and p53 positivity. There was also no relation between the MVD and p53 expression. MVD and p53 expression could not predict survival in these patients with pancreatic adenocarcinoma. There was no correlation with p53 expression and intratumoral microvessel density. High MVD was associated with tumor size grater than 3,0 cm and positive margins

As células tumorais prosperam como consequência da capacidade de resistir aos mecanismos de morte celular e de evasão da vigilância imunológica. Nós propomos que, em cânceres que possuem o supressor de tumor p53 selvagem, a remediação de ambas dessas defesas pode ser promovida pela transferência genica combinada de vetores adenovirais portadores dos transgenes de p19Arf (proteína supressora de tumor, parceira funcional de p53) e de interferon-beta (IFNbeta, citocina imunomoduladora). De fato, em resultados anteriores, notamos que a transdução combinada (p19Arf/IFNbeta), mas não os tratamentos individuais, em células de melanoma murino B16F10 resulta em aumento massivo de morte celular. Porém a capacidade destas células em processo de morte de desencadear imunidade antitumoral não foi analisada. Nesta tese e em estudos complementares, buscamos investigar os mecanismos moleculares de morte celular envolvidos na resposta imune estimulada por p19Arf/IFNbeta e explorar sua aplicação como imunoterapia do câncer. Inicialmente, em modelo de vacinação profilática, revelamos que o tratamento combinado em células B16F10 promove a expressão de IL-15, ULBP1, dos receptores de morte FAS/APO1 e KILLER/DR5, assim como uma resposta de células natural killer que rejeitam estas células tratadas quando inoculadas em camundongos imunocompetentes singênicos. Após desafio tumoral no flanco oposto, a progressão desses tumores foi fortemente reduzida devido ao engajamento de linfócitos T CD4+ e CD8+, que apresentaram produção aumentada das citocinas IFN-? e TNF-alfa e medeiam proteção antitumoral de longo prazo. Em seguida, explorando um contexto de imunização diferente, a transferência de gênica in situ foi realizada em carcinoma heterotópico de pulmão e exibiu proteção significativa contra um desafio tumoral secundário, apenas quando o tumor primário foi tratado com p19Arf/IFNbeta. Análise de transcriptoma destes tumores indicou uma assinatura quimiotáxica de neutrófilos e linfócitos T CD8+ através das quimiocinas CCL3, CXCL3 e da IL-1beta. Em apoio destas observações, análises mecanicistas in vitro revelaram que células tratadas com p19Arf/IFNbeta ativam programas apoptóticos de p53 e antivirais de IFNbeta, enquanto sucumbem a um processo de morte por necroptose que também libera moléculas de morte celular imunogênica (MCI), calreticulina, ATP e HMGB1. No entanto, procurando potencializar ainda mais o benefício terapêutico dos nossos vetores, exploramos sua associação com o quimioterápico imunogênico doxorrubicina (Dox), que também é indutor de MCI. E nesta associação, percebemos que a Dox aumenta não apenas os níveis de morte celular, mas também a imunogenicidade das células tratadas, proporcionando em um modelo de vacina terapêutica, um controle tumoral superior em camundongos que já portavam antes da vacinação tumores B16F10 ou MCA205. Além disso, a associação in situ destas terapias restaurou a eficácia de uma dose sub-terapêutica de Dox, que em contraste com sua dose terapêutica, não prejudica a função cardíaca. Finalmente, também exploramos a associação com o bloqueio dos pontos de controle imunológicos PD-1 ou CTLA-4, que no modelo de vacina terapêutica, sua associação induziu maior rejeição completa de tumores B16F10. Em conclusão, aqui apresentamos evidências sobre a capacidade da combinação p19Arf/IFNbeta de induzir morte celular e estimulação imunológica. E ressaltamos seu potencial como uma estratégia de imunoterapia do câncer / Cancer cells thrive as a consequence of resisting cell death mechanisms and escaping from immune surveillance. We propose that, in cancers that harbor the wild-type tumor suppressor p53, remediation of both of these defenses can be achieved by harnessing the adenoviral vector mediated gene transfer of p19Arf (tumor suppressor protein, p53 functional partner) together with interferon-beta (IFNbeta, immunomodulatory cytokine). Indeed, in our initial observations, it was noticed that combined-transduction (p19Arf/IFNbeta), but not the individual treatments, of B16F10 mouse melanoma cells results in massive cell death levels. Yet, the capability of these dying cells to unleash antitumor immunity was not investigated. Here in this thesis and in complementary studies, we sought to investigate the molecular mechanisms of cell death involved in the p19Arf/IFNbeta immune stimulation and explore its potential as a mediator of cancer immunotherapy. First, in a prophylactic B16F10 vaccine model, we revealed that the dual treatment led to the up-regulation of IL-15, ULBP1, FAS/APO1 and KILLER/DR5 death receptors, plus a natural killer cell response that completely rejects treated cells when inoculated in syngeneic immunocompetent mice. Whereas, upon a contralateral tumor challenge, progression was strongly reduced by engaging both CD4+ and CD8+ T cells, which displayed augmented production of IFN-? and TNF-alpha cytokines and provided long term antitumor protection. Next, exploring different immunization context, in situ gene transfer in a heterotopic lung carcinoma exhibited significant protection against a secondary tumor challenge only when the primary tumor was treated with p19Arf/IFNbeta. Transcriptome analysis of these treated tumors indicated a chemotaxic signature of neutrophils and CD8+ T cells with the involvement of CCL3, CXCL3 chemokines and IL-1beta. Moreover, in support of this evidence, mechanistic in vitro studies revealed that p19Arf/IFNbeta treated cells reactivate p53 apoptotic and IFNbeta antiviral programs, while succumbing to a necroptosis cell death processes that also releases immunogenic cell death (ICD) molecules, calreticulin, ATP and HMGB1. Yet, aiming to potentiate therapeutic benefit of our vectors, we explored their association with doxorubicin (Dox) immunogenic chemotherapy, which is also an inducer of ICD. And in this setting, this association with Dox enhances not only cell death levels but also immunogenicity of treated cells, providing superior tumor control in a therapeutic vaccine model, where mice were already bearing B16F10 tumors or MCA205 sarcomas before vaccination. Moreover, associated use of these therapies in situ rescued efficacy of a sub-therapeutic dose of Dox, which in contrast to its therapeutic dose, does not impair cardiac function. Finally, we also evaluated the association with PD-1 or CTLA-4 checkpoint blockade immunotherapy, which in the therapeutic vaccine model induced full tumor rejection in a greater number of mice. In sum, here we provide compelling evidence for the ability of the p19Arf/IFNbeta combined gene transfer to promote cell death and immunogenic stimuli and underscored its potential to be applied as a cancer immunotherapy strategy

O carcinoma epidermoide do esôfago continua sendo a principal neoplasia maligna esofágica na população brasileira. Os objetivos desta investigação foram: avaliar a imuno-expressão de um grupo de proteínas relacionadas à via intrínseca da apoptose (bax, APAF-1 e citocromo c) e da proteína p53 em um grupo de carcinomas epidermoides do esôfago; confrontar estes resultados com a atividade proliferativa medida pela imuno-expressão do antígeno Ki67 e com a atividade apoptótica medida pela imuno-expressão da caspase 3 clivada; e confrontá-los com parâmetros implicados no estadiamento do carcinoma epidermoide do esôfago (invasão local ou pT, estado dos linfonodos regionais ou pN, grau de diferenciação do tumor primário e local do tumor primário no esôfago) e com o tamanho do tumor primário. De um grupo inicial de 91 carcinomas esofágicos consecutivos, 66 carcinomas epidermoides do esôfago foram revistos, alocados em micromatrizes teciduais e submetidos à técnica de imuno-peroxidase com anticorpos primários anti: bax, APAF-1, citocromo c, p53, Ki67 e caspase 3 clivada. Suas imuno-expressões foram semiquantificada de 0 a 5+, exceto caspase 3 clivada que foi contada em 1000 células. Apresentaram amostras válidas um conjunto de 63 carcinomas epidermoides do esôfago. A mediana de imuno-expressão destas 6 proteínas foi: 2+, 5+, 5+, 5+, 3+ e 26, respectivamente. Houve correlação positiva entre a imunoexpressão do antígeno Ki67 e a de caspase 3 clivada (coeficiente rho de Spearman =0,373, p=0,003). Houve associação entre a imunoexpressão de APAF-1 e o grau de diferenciação, com valores maiores de APAF-1 para os carcinomas epidermoides do esôfago bem diferenciados (mediana de 5+ para tumores bem diferenciados, contra mediana de 2+ para tumores pouco diferenciados, p<0,001, teste de Kruskal-Wallis). Houve associação entre o tamanho do tumor primário e o nível de invasão local do tumor primário, com tamanhos maiores quanto maior o nível de invasão local dos carcinomas epidermoides do esôfago (mediana de 32,5 mm para os tumores pT1 e mediana de 50,0 mm para os tumores pT3 ou pT4, p=0,027, teste de Kruskal-Wallis). Não houve associação entre as demais variáveis. Embora atividade proliferativa e atividade apoptótica caminhem juntas nos carcinomas epidermoides do esôfago, no estágio invasivo do principal tipo histológico de carcinoma esofágico da população brasileira, não são mais os fatores ligados à via intrínseca da apoptose que influenciam a sua progressão. Além disso, se a imunoexpressão aumentada da proteína APAF-1 estimula a diferenciação nos carcinomas epidermoides esofágicos, não o faz através de estímulo da atividade apoptótica pura e simplesmente / Squamous cell carcinoma of the esophagus remains as the major malignant esophageal neoplasm in the Brazilian population. The objectives of this study were: to assess the immunoexpression of a group of proteins related to the intrinsic pathway of apoptosis (bax, APAF-1 and cytochrome c) and to p53 protein in squamous cell carcinoma of the esophagus; to confront these results with proliferative activity measured by the immunoexpression of Ki67 and with apoptotic activity measured by the immunoexpression of cleaved caspase 3; and to confront them with parameters involved in the staging of squamous cell carcinoma of the esophagus(local invasion or pT, lymph node status or pN, grade of differentiation of primary tumor and site of primary tumor in the esophagus) and with size of primary tumor. From a starting group of 91 consecutive carcinomas of the esophagus, 66 squamous cell carcinomas of the esophagus were selected, revised, placed in tissue microarrays blocks, and submitted to immunoperoxidase technique with primary antibodies to: bax, APAF-1, cytochrome c, p53, Ki67 and cleaved caspase 3. The immunoexpression was semiquantified in a scale from 0 to 5+, except for cleaved caspase 3, whicht was counted in 1000 cells. Sixty three squamous cell carcinomas of the esophagus displayed valid cores for analysis. The median immunoexpression of these 6 proteins were: 2+, 5+, 5+, 5+, 3+ and 26, respectively. A positive correlation was found between Ki67 antigen and cleaved caspase 3 immunoexpression (Spearmans rho coefficient =0.373, p=0.003). There was association between the immunoexpression of APAF-1 and the grade of differentiation, with higher values of APAF-1 for well differentiated squamous cell carcinomas of the esophagus (median of 5+ for well differentiated tumors and median of 2+ for poorly differentiated tumors, p<0.001, Kruskal-Wallis test). The size of primary tumor was statistically associated to the degree of local invasion of primary tumor, with higher size associated to deeper local invasion (median of 32.5 mm for pT1 tumors and median of 50.0 mm for pT3 or pT4 tumors, p=0.027, Kruskal-Wallis test). There was no association among the other variables. Although proliferative activity and apoptotic activity go together in squamous cell carcinomas of the esophagus, the factors involved in the intrinsic pathway of apoptosis does not differ significantly according to the histological parameters in the invasive phase of the development of squamous cell carcinoma of esophagus. Moreover, , if increased immunoexpression of APAF-1 stimulates differentiation of squamous cell carcinomas of the esophagus, it does not work through direct higher apoptotic activity