Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 83-98.

Cancer is the second leading cause of death in the United States, and it results from genetic alterations that promote the survival and proliferation of neoplastic cells. One of the most commonly disrupted cancer gene networks is the ARF-Mdm2-Tip60-p53 pathway. Inactivation of the ARF, Tip60 and p53 tumor suppressors and/or overexpression of the Mdm2 oncogene occurs in most, if not all, human cancers. An improved molecular understanding of that pathway, especially how it becomes activated, is expected to advance the development of innovative therapeutics aimed at restoring its function in tumors. Our group originally discovered NIAM (Nuclear Interactor of ARF and Mdm2) as a novel binding partner of ARF that has several functional ties to Mdm2 and p53. Early studies showed that NIAM is negatively regulated by Mdm2, can collaborate with ARF to block cell proliferation, and is a new activator of p53-mediated transcription. NIAM could also act independent of those factors to suppress cell proliferation and promote chromosomal stability, and microarray studies suggested its expression is significantly reduced in many human cancers. Those findings led us to speculate that NIAM is a tumor suppressor that functions in both the ARF-Mdm2-p53 pathway as well as other undefined anti-cancer pathways. My thesis research explored two different aspects of that hypothesis: 1) how does NIAM activate p53, and 2) is NIAM a tumor suppressor? Initial work showed that NIAM could stimulate p53 independently of ARF, the major activator of p53, indicating that other factors must be required. My studies revealed that NIAM indirectly promotes p53 activation through functional interactions with two other p53 regulators, Tip60 and Mdm2. Tip60 is an acetyltransferase that activates p53 through direct association on p53 target promoters as well as acetylation of p53 at lysine 120 (K120). I found that NIAM can induce K120 acetylation of p53; however, NIAM's association with Tip60 (not the acetylation of p53) was essential for maximal p53 transcriptional activation. Mdm2, the major antagonist of p53, is an E3 ubiquitin ligase that promotes p53 ubiquitylation and degradation. I found that NIAM disrupts Mdm2-p53 complexes and blocks p53 ubiquitylation, thereby interfering with p53 inhibition by Mdm2. Thus, NIAM regulates two critical pathways that control p53 function and are altered in human cancers, implying an important role for NIAM in tumorigenesis. To test that idea directly, we generated NIAM gene-trap knockout mice (hypomorphs) that expressed greatly reduced yet detectable levels of NIAM in all tissues. Fifty percent of the NIAM knockout mice developed benign and early stage cancers, including B-cell lymphoma, whereas all age-matched control animals were tumor-free. These results showed that NIAM loss causes increased susceptibility to tumorigenesis. In sum, my work suggests a significant role for NIAM in p53 control and tumor biology. Additional studies will be needed to determine which physiological or pathological signals normally engage NIAM to promote p53 function and suppress tumor development. It is anticipated that the NIAM knockout mice will provide an outstanding platform to interrogate NIAM's biological role in cancer, particularly in ARF-Mdm2-Tip60-p53 signaling as well as other pathways affecting genome maintenance. Ultimately, insights gained from such studies may justify novel therapies that seek to restore NIAM activity in tumors.

In cells undergoing stress, the p53 transcription factor is stabilized and activates the expression of numerous genes contributing to p53-mediated tumor suppression. One p53 target gene is Mdm2, which encodes an oncoprotein that binds and ubiquitinates p53 for proteasomal degradation, thus limiting the amplitude and duration of the p53-mediated stress response. Our lab recently discovered that Mdm2 phosphorylation by ATM and c-Abl regulates the DNA damage response and tumorigenesis in mice. AKT has also been found in transfection studies to phosphorylate Mdm2 at serine residues 166 and 186 (mouse S163 and S183) to alter p53 activity. However, the physiological significance of Mdm2 phosphorylation by Akt remains unknown. Therefore, I generated Mdm2S163A or Mdm2S183A mice expressing mutant Mdm2 incapable of being phosphorylated by Akt. In contrast with our previous studies, Akt phosphorylation of Mdm2 does not alter spontaneous tumorigenesis or the DNA damage response to ionizing radiation. However, Akt phosphorylation of Mdm2-S183 (but not -S163) upregulates nuclear localization of Mdm2, destabilizes p53, and reduces p53-mediated senescence in response to elevated levels of reactive oxygen species (ROS). To examine the effects of Mdm2-S183 phosphorylation on p53 tumor suppression, I utilized three different mouse models of ROS-induced cancer. Increased levels of p53 and senescence in Mdm2S183A mice yielded reduced tumorigenesis in an activated Ras model of lung cancer, a phorbal ester-induced skin cancer model, and a diethylnitrosamine-induced model of hepatocellular carcinoma. Since AKT is also important regulator of cell metabolism, I explored the impact of the Mdm2-S183 allele on metabolic functions. Mdm2 phosphorylation by Akt reduced glucose metabolism via glycolysis in vitro, and reduced insulin tolerance in mice, without altering glucose tolerance and glucose-stimulated insulin secretion. Collectively, these findings document a unique physiologic role for the AKT-Mdm2-p53 signaling axis in regulating cell growth and tumorigenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcription factor and tumor suppressor protein p53 critically regulates cell survival or death in response to cellular stress. p53 can activate genes involved in a wide variety of processes, including apoptosis, cell cycle arrest, angiogenesis, metabolism, and senescence. Mutations in p53 are common in cancer and alter its interactions with other proteins, but there are other mechanisms and posttranslational modifications that can alter these interactions as well. In some tumors, such as renal cell carcinoma, p53 is commonly inactive even though mutations to TP53 are rare. This suggests that there are other biochemical mechanisms of inhibition, which we explore in this study. Mutations in the DNA-binding domain of p53 result in conformational changes that enable p53 to interact with and inhibit its family member p73, thereby promoting cell survival instead of apoptosis. In contrast, it has been reported that wild-type p53 does not bind to p73. We found that JNK-mediated phosphorylation of Thr81 in the proline-rich domain (PRD) of p53 enabled wild-type p53 to form a complex with p73. The dimerization of wild-type p53 with p73 facilitated the expression of apoptotic target genes such as PUMA and BAX, as well as the induction of apoptosis. In addition to the apoptotic function of p53, the tumor suppressor also plays a major role in the inhibition of angiogenesis. Here we also report a new mechanism where the Mdm2 oncoprotein can indirectly inactive p53 through the regulation of the tumor suppressor VHL. In response to hypoxia, VHL can bind p53, which results in activation of several anti-angiogenic targets of p53 such as THBS1 and COL18A1. Mdm2 regulates the VHL-p53 interaction by conjugating nedd8 to VHL within a region that is important for the VHL-p53 interaction, blocking the induction of anti-angiogenic genes and resulting in a proangiogenic phenotype. Due to its positive regulation of major proangiogenic proteins and its negative regulation of potent inhibitors of angiogenesis, we propose that the oncoprotein Mdm2 is the angiogenic switch. These findings refine our understanding of p53 interactions and activation, specifically for p53-p73 induced cell death and p53-VHL inhibition of angiogenesis. / 2020-08-05

No description available.

O mecanismo de oncogênese na laringe pode ser controlado por vários fatores, entre eles fatores envolvidos na regulação do ciclo celular e outros de risco, tais como exposição prolongada ao fumo e álcool. O desenvolvimento do câncer de laringe também pode estar associado à infecção pelo HPV. Este estudo, análise imunoistoquímica quantitativa de p53, p27 e Mdm2, foi realizado em 54 pacientes com carcinoma invasivo de laringe subdivididos em: carcinoma sem metástase (laryngeal squamous cell carcinoma without metastasis - LSCCWT), com metástase (laryngeal squamous cell carcinoma with metastasis - LSCCW) e linfonodos cervicais (limph nodes biopsies - LB). A detecção e tipificação do HPV foram realizadas pela reação em cadeia da polimerase (PCR) e os tipos de HPV avaliados foram HPV 6, 11, 16, 18, 31 e 33. Na análise quantitativa, alta expressão de p53, p27 e Mdm2 foi observada nos grupos LSCCW e LSCCWT assim como nas biópsias dos linfonodos cervicais, indicando que a avaliação dessas proteínas poderia não discriminar carcinomas de laringe metastáticos e não-metastáticos. Detecção do HPV foi verificada em apenas 7.4% dos casos. Dentre os pacientes HPV positivos, verificou-se expressão negativa de p53. Por outro lado, alta expressão de p27 e Mdm2 foi observada. Em conclusão, a avaliação quantitativa de p53, p27 e Mdm2 não permite traçar um perfil complementar em lesões metastáticas de laringe. / The mechanism of larynx oncogenesis could be controlled by various factors, most of them involved in cell cycle regulation and other risk factors such as smoking and alcohol abuse. The development of laryngeal carcinoma is associated with human papillomavirus (HPV) infection. In this study, quantitative immunohistochemistry was perfomed for p53, p27 and Mdm2 in 54 patients with invasive laryngeal squamous cell carcinoma without metastasis (LSCCWT), with metastasis (LSCCW) and cervical lymph nodes (LB). HPV detection and typing was performed by PCR and the HPV types evaluated were HPV 6, 11, 16, 18, 31 and 33. In the quantitative analysis higher p53, p27 and Mdm2 expression was observed in both LSCCW and LSCCWT, as well in cervical lymph node biopsies with metastasis, may indicating that evaluation of these proteins may not discriminate between metastatic and non-metastatic laryngeal carcinoma. HPV was found in 7.4% of the cases. Among HPV- positive patients, p53 expression was negative. On the other hand, high p27 and Mdm2 expression was observed. In conclusion, these data suggest that quantitative evaluation of p53, p27 and Mdm2 does not permit to determine a complementary profile in metastatic laryngeal lesions.

A proteína P53 pode estar virtualmente alterada em todos os cânceres humanos e portanto, na ausência de mutação, uma possibilidade para a inativação da p53 é a formação de complexo com outras proteínas, tal como a proteína Mdm2. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Mdm2 na ausência de expressão da proteína P53 em adenomas pleomórficos. O objetivo deste estudo foi analisar a expressão das proteínas Mdm2, P53, P21 e Akt em adenoma pleomórfico e o mioepitelioma através das técnicas de imunoistoquímica, western blotting e imunofluorescência. A superexpressão de Mdm2 e Akt foi encontrada na maioria das linhagens e lesões utilizadas neste estudo enquanto as proteínas P53 e P21 não demonstraram expressão nas neoplasias estudadas. As superexpressões das proteínas Mdm2 e Akt estão relacionadas à tumorigênese em neoplasias benignas de glândula salivar. / The P53 protein can be altered in virtually all human cancers and in the absence of mutations, P53 inactivation is possible via complex formation with others proteins, such as the Mdm2. Previous studies from our laboratory showed overexpression of mdm2 and lack of p53 expression in pleomorphic adenomas. The aim of this study was to analyze the expression of Mdm2, P53, P21 and Akt proteins in pleomorphic adenomas and myoepiteliomas by Western blotting, immunohistochemistry and immunofluorescence techniques. Overexpression of Mdm2 and Akt was present in the majority of cell lineages and tumors studied, while the expression of P53 and P21 proteins was considered absent. Overexpression of Mdm2 and Akt are related to the tumorigenesis of benign salivary gland neoplasms.

A proteína Akt modula a função de numerosos substratos envolvidos na regulação da sobrevivência celular, progressão do ciclo celular e crescimento celular. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Akt em adenoma pleomórfico, mioepitelioma e carcinoma adenóide cístico. O objetivo deste estudo foi analisar a via da proteína Akt através da avaliação da expressão das proteínas NFkB e PTEN em neoplasias benignas e malignas de glândulas salivares através das técnicas de imunohistoquímica, western blotting e imunofluorescência, e a possível interação protéica direta entre p-Akt/Mdm2 e p-Akt/PTEN em linhagem de carcinoma adenóide cístico. A superexpressão nuclear na proteína PTEN foi encontrada nas duas neoplasias malignas estudadas. Além disso, não foi observada interação direta entre as proteínas p-Akt/Mdm2 e p-Akt/PTEN, as quais apresentam localização nuclear em neoplasias de glândulas salivares. / The Akt protein modulates the function of numerous substrates involved in the regulation of cell survival, cell cycle progression and cell growth. Previous studies from our laboratories showed overexpression of Akt and Mdm2 followed by the lack of p53 expression in pleomorphic adenoma, myoepithelioma and adenoid cystic carcinoma. The aim of this study was to analyze the Akt pathway through evaluation of expression of NFkB and PTEN proteins in pleomorphic adenoma, carcinoma ex pleomophic adenoma and adenoid cystic carcinoma by western blotting, immunofluorescence and immunohistochemical techniques, and we have intended to analyse a possible direct interaction between p-Akt/Mdm2 and p-Akt/ PTEN protein in salivary gland tumours. Overexpression of nuclear PTEN was present in both carcinomas studied. In addition, there was no direct interaction between p-Akt/Mdm2 and p-Akt/ PTEN protein, which presents a nuclear localization in salivary gland tumours.

Les résultats insuffisants de la radiochimiothérapie conventionnelle ont motivé l’évaluation de nouvelles cibles afin de moduler la radiosensibilité tumorale: voies intrinsèques impliquées dans la réponse aux rayonnements ionisants, vascularisation tumorale, stroma non vasculaire. A travers cette thèse, nous avons évalué trois nouvelles stratégies de radiosensibilisation pharmacologique. Nous avons d’abord étudié en association à la radiothérapie l’intérêt de la modulation de l’axe p53/Mdm2 par le JNJ26854165, un inhibiteur de la dégradation de p53 par le protéasome. Les résultats in vitro et in vivo dans des xénogreffes sous-cutanées de cancers bronchiques non à petites cellules (CBNPC) montrent que cette stratégie permet d’améliorer significativement l’efficacité de la radiothérapie. Nous avons également rapporté des résultats encourageants in vitro dans plusieurs lignées cellulaires tumorales avec un nouvel agent antivasculaire ciblant la tubuline, l’EHT 6706. Cette stratégie augmentait l’efficacité de l’irradiation et potentialisait l’effet antiprolifératif de certains agents de chimiothérapie conventionnelle. Enfin, le développement le plus abouti a consisté en l’évaluation de l’association d’un triple inhibiteur de MET/AXL/FGFR en association à l’irradiation in vitro et dans des modèles de CBNPC implantés en xénogreffes sous-cutanées, mais également sous forme de tumeurs pulmonaires orthotopiques. Cet agent pharmacologique potentialisait l’efficacité de la radiothérapie dans des lignées ne surexprimant pas MET. Il est apparu que l’activité de la drogue faisait intervenir, au moins partiellement, l’inhibition de l’activité d’acteurs de la cytocinèse. Ces trois évaluations, qui s’inscrivent dans la recherche translationnelle, montrent l’importance de la recherche préclinique pour les études d’association aux rayonnements ionisants. Seul un développement préclinique rationnel permettra de faire émerger de nouveaux standards dans le domaine de la biomodulation pharmacologique de la radiosensibilité tumorale. / Insufficient results of conventional chemoradiation have encouraged assessment of new targets for radiosensitization: intrinsic cellular pathways involved in radiation response, tumor angiogenesis, and nonvascular stroma. We have investigated these three strategies for pharmacological radiosensitization. First, we examined the usefulness of targeting p53/Mdm2 pathway in combination with irradiation. In vitro and in vivo results obtained in non-small cell lung carcinoma (NCSLC) showed that this strategy was promising for enhancing radiation efficacy. We also found encouraging results within several cell lines with a novel vascular disrupting agent targeting tubulin. This strategy enhanced radiation effects and also increased the antiproliferative effects of various chemotherapeutics. Finally, the most advanced preclinical development was obtained with a novel MET/AXL/FGFR inhibitor, which improved effectiveness of radiation therapy in vitro and in subcutaneous and orthotopic models of non MET-dependent cell cancer lines. This effect was not only related to an inhibition of stroma/cancer cell interactions, as it probably involved activity toward actors of cytocinesis. These studies, which are part of translational research, highlight the importance of preclinical investigations in the area of radiation research. Only rationale preclinical development will allow new standards to emerge for pharmacological modulation of tumor radiosensitivity.

A proteína P53 pode estar virtualmente alterada em todos os cânceres humanos e portanto, na ausência de mutação, uma possibilidade para a inativação da p53 é a formação de complexo com outras proteínas, tal como a proteína Mdm2. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Mdm2 na ausência de expressão da proteína P53 em adenomas pleomórficos. O objetivo deste estudo foi analisar a expressão das proteínas Mdm2, P53, P21 e Akt em adenoma pleomórfico e o mioepitelioma através das técnicas de imunoistoquímica, western blotting e imunofluorescência. A superexpressão de Mdm2 e Akt foi encontrada na maioria das linhagens e lesões utilizadas neste estudo enquanto as proteínas P53 e P21 não demonstraram expressão nas neoplasias estudadas. As superexpressões das proteínas Mdm2 e Akt estão relacionadas à tumorigênese em neoplasias benignas de glândula salivar. / The P53 protein can be altered in virtually all human cancers and in the absence of mutations, P53 inactivation is possible via complex formation with others proteins, such as the Mdm2. Previous studies from our laboratory showed overexpression of mdm2 and lack of p53 expression in pleomorphic adenomas. The aim of this study was to analyze the expression of Mdm2, P53, P21 and Akt proteins in pleomorphic adenomas and myoepiteliomas by Western blotting, immunohistochemistry and immunofluorescence techniques. Overexpression of Mdm2 and Akt was present in the majority of cell lineages and tumors studied, while the expression of P53 and P21 proteins was considered absent. Overexpression of Mdm2 and Akt are related to the tumorigenesis of benign salivary gland neoplasms.