p53, known as the “guardian of the genome”, is the most well-characterized tumor suppressor gene. The central role of p53 is to prevent genome instability. p53 is the central node in an incredibly elaborate genome defense network for receiving various input stress signals and controlling diverse cellular responses. The final output of this network is determined not only by the p53 protein itself, but also by other p53 cooperating proteins. p300 and CBP (CREB-Binding Protein) act as multifunctional regulators of p53 via acetylase and ubiquitin ligase activities. Prior work in vitro has shown that the N-terminal 595 aa of p300 encode both generic ubiquitin ligase (E3) and p53-directed E4 functions. Analysis of p300 or CBP-deficient cells revealed that both coactivators were required for endogenous p53 polyubiquitination and the normally rapid turnover of p53 in unstressed cells. Unexpectedly, p300/CBP ubiquitin ligase activities were absent in nuclear extracts and exclusively cytoplasmic. In the nucleus, CBP and p300 exhibited differential regulation of p53 gene target expression, C-terminal acetylation, and biologic response after DNA damage. p300 activated, and CBP repressed, PUMA expression, correlating with activating acetylation of p53 C-terminal lysines by p300, and a repressive acetylation of p53 lysine-320 induced by CBP. Consistent with their gene expression effects, CBP deficiency augmented, and p300 deficiency blocked, apoptosis after doxorubicin treatment. Subcellular compartmentalization of p300/CBP’s ubiquitination and transcription activities reconciles seemingly opposed functions—cytoplasmic p300/CBP E4 activities ubiquitinate and destabilize p53, while nuclear p300/CBP direct p53 acetylation, target gene activation, and biological outcome after genotoxic stress. p53 is a prominent tumor suppressor gene and it is mutated in more than 50% of human tumors. Reactivation of endogenous p53 is one therapeutic avenue to stop cancer cell growth. In this thesis, we have identified S5as a critical regulator of p53 degradation and activity. S5a is a non-ATPase subunit in the 19S regulatory particle of the 26S proteasome. Our preliminary data indicates that S5a is required for p53 instability and is a negative regulator of p53 tranactivation. As a negative regulator of p53, S5a may therefore also represent a new target for cancer drug development against tumors that specifically maintain wild type p53.

The p53 transcription factor responds to various cellular stressors by regulating the expression of numerous target genes involved in cellular processes such as cell cycle arrest, apoptosis, and senescence. As these downstream pathways are harmful to the growth and development of normal cells when prolonged or deregulated, p53 activity needs to be under tight regulatory control. The Mdm2 oncoprotein is the chief negative regulator of p53, and many mouse models have demonstrated that absence of Mdm2 expression leads to constitutive p53 activation in a variety of cell types. While unregulated p53 can be deleterious to cells, functional p53 is essential for tumor suppression, as many human cancers harbor p53 mutations and p53 knockout mice rapidly develop spontaneous tumors. Therefore, the mechanisms that control p53 regulation by Mdm2 are critical to ensure p53 activity in the appropriate cellular context. Many genetically engineered mouse models have been created to analyze p53 and Mdm2 functions and these studies have yielded valuable insights into their physiological roles. This dissertation will describe the generation and characterization of novel mutant Mdm2 mouse models and their use to interrogate the roles of p53-Mdm2 signaling in tissue homeostasis and cell stress responses. Deletion of Mdm2 in epidermal progenitor cells of the skin and hair follicles resulted in progressive hair loss and decreased skin integrity, phenotypes that are characteristic of premature aging. Furthermore, p53 protein levels, p53 target gene expression, and cellular senescence were all upregulated in the skins of these mice, and epidermal stem cell numbers and function were diminished. These results indicate that Mdm2 is necessary to limit p53 activity in adult tissues to ensure normal stem cell function. Additional mouse models used to determine the role of Mdm2 phosphorylation will also be presented. DNA damage triggers an extensive cellular response, including activation of the ATM kinase. ATM activity is necessary for p53 protein stabilization and, therefore, p53 activation, but in vivo evidence suggests that phosphorylation of p53 itself had little effect on p53 stability. ATM was previously shown to phosphorylate MDM2 at serine residue 395 (394 in mice), and we generated knock-in mutant mouse models to study the role of this posttranslational modification in vivo. Absence of this phosphorylation site led to greatly diminished p53 stability and function in response to γ-irradiation and increased spontaneous tumorigenesis in mice. Conversely, a phosphomimic model demonstrated prolonged p53 activation in cells treated with γ-irradiation, which revealed that phosphorylation of this Mdm2 residue controls the duration of the DNA damage response. Therefore, these mouse models have uncovered new roles for the p53-Mdm2 regulatory axis in vivo and will be useful reagents in future studies of posttranslational modifications in oncogene and DNA damage-induced tumorigenesis.

Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancer-related death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of Krüppel-like factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of Krüppel-like factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. Single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6-mediated tumor and metastasis suppression, we performed gene expression profiling and ChIP-sequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6-mediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gain-of- function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gain-of-function properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformation-associated phenotypes in these cells. However, p53R172H-expressing mice did not have enhanced tumor formation or metastasis compared to p53-null mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gain-of-function mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gain-of-function effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53-null and p53R172H-expressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients.

Animals have evolved complicated and overlapping mechanisms to guard against the development of cancer and infection by pathogenic organisms. ARF, a potent tumor suppressor, positively regulates p53 by antagonizing p53’s negative regulator, MDM2, which in turn results in either apoptosis or cell cycle arrest. ARF also has p53-independent tumor suppressor activity. The CtBP transcriptional co-repressors promote cancer cell survival and migration/invasion. CtBP senses cellular metabolism via a regulatory dehydrogenase domain, and is a target for negative regulation by ARF. ARF targets CtBP to the proteasome for degradation, which results in the up regulation of proapoptotic BH3-only proteins, and p53-independent apoptosis. CtBP inhibition by ARF also up regulates PTEN, reducing cancer cell motility, making CtBP a potential therapeutic target in human cancer. The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and is cytotoxic to cancer cells from a wide variety of tissues. MTOB induced apoptosis was independent of p53, and correlated with the de-repression of the pro-apoptotic CtBP repression target Bik. CtBP over-expression, or Bik silencing, rescued MTOB-induced cell death. MTOB did not induce apoptosis in mouse embryonic fibroblasts (MEFs), but was increasingly cytotoxic to immortalized and transformed MEFs, suggesting that CtBP inhibition may provide a suitable therapeutic index for cancer therapy. In human colon cancer cell peritoneal xenografts, MTOB treatment decreased tumor burden, and induced tumor cell apoptosis. To verify the potential utility of CtBP as a therapeutic target in human cancer the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Targeting CtBP with a small molecule like MTOB may thus represent a useful and widely applicable therapeutic strategy in human malignancies. ARF has long been known to respond to virally encoded oncogenes. Recently, p14ARF was linked to the innate immune response to non-transforming viruses in mice. Therefore a wider role for the ARF pathway in viral infection was considered. Previous studies linking p53 to multiple points of the Human Immunodeficiency Virus-1 (HIV-1) life cycle suggested that ARF may also play a role in the HIV life cycle. In this study the interdependency of ARF and HIV infection was investigated. ARF expression was determined for a variety of cell types upon HIV infection. In every case, ARF levels exhibited dynamic changes upon HIV infection-in most cases ARF levels were reduced in infected cells. The impact of ARF over-expression or silencing by RNAi on HIV infection was also examined. Consistently, p24 levels were increased with ARF overexpression, and decreased when ARF was silenced. Thus ARF and HIV modulate each other, and ARF may paradoxically play a positive role in the HIV life cycle.

Yes / The NAD(+)-dependent deacetylase SIRT1 is involved in diverse cellular processes, and has also been linked with multiple disease states. Among these, SIRT1 expression negatively correlates with cancer survival in both laboratory and clinical studies. Active regulator of SIRT1 (AROS) was the first reported post-transcriptional regulator of SIRT1 activity, enhancing SIRT1-mediated deacetylation and downregulation of the SIRT1 target p53. However, little is known regarding the role of AROS in regulation of SIRT1 during disease. Here, we report the cellular and molecular effects of RNAi-mediated AROS suppression, comparing this with the role of SIRT1 in a panel of human cell lines of both cancerous and non-cancerous origins. Unexpectedly, AROS is found to vary in its modulation of p53 acetylation according to cell context. AROS suppresses p53 acetylation only following the application of cell damaging stress, whereas SIRT1 suppresses p53 under all conditions analysed. This supplements the original characterization of AROS but indicates that SIRT1 activity can persist following suppression of AROS. We also demonstrate that knockdown of AROS induces apoptosis in three cancer cell lines, independent of p53 activation. Importantly, AROS is not required for the viability of three non-cancer cell lines indicating a putative role for AROS in specifically promoting cancer cell survival.

No / Zinc oxide (ZnO) nanoparticles are the mostly used engineered metal oxide nanoparticles in consumer products. This has increased the likelihood of human exposure to this engineered nanoparticle (ENPs) through different routes. At present, the majority of the studies concerning ZnO ENPs toxicity have been conducted using in vitro and in vivo systems. In this study, for the first time we assessed the effect of ZnO ENPs on the major cellular pathways in the lymphocytes of healthy individuals as well as in susceptible patients suffering from lung cancer, chronic obstructive pulmonary disease (COPD) and asthma. Using the differential expression analysis, we observed a significant (P < 0.05) dose-dependent (10, 20 and 40 microg/ml for 6h) increase in the expression of tumour suppressor protein p53 (40, 60 and 110%); Ras p21 (30, 52 and 80%); c-Jun N-terminal kinases; JNKs) (28, 47 and 78%) in lung cancer patient samples treated with ZnO ENPs compared to healthy controls. A similar trend was also seen in COPD patient samples where a significant (P < 0.05) dose-dependent increase in the expression of tumour suppressor protein p53 (26, 45 and 84%), Ras p21 (21, 40 and 77%), JNKs (17, 32 and 69%) was observed after 6h of ZnO ENPs treatment at the aforesaid concentrations. However, the increase in the expression profile of tested protein was not significant in the asthma patients as compared to controls. Our results reiterate the concern about the safety of ZnO ENPs in consumer products and suggest the need for a complete risk assessment of any new ENPs before its use.

Le gène suppresseur de tumeurs TP53 exprime plusieurs isoformes, dont Δ40p53 (perte du domaine de transactivation) et Δ133p53 (perte du domaine de transactivation et d’une partie du domaine de liaison à l’ADN). Ces isoformes inhibent l’activité suppressive de p53 et seraient sur-exprimées dans les cancers (sein et mélanome). Dans les cancers faiblement associés à une mutation TP53, ces isoformes seraient de bons candidats pour inactiver p53. Il convient de comprendre les mécanismes transcriptionnels qui régulent leurs expressions. Δ133p53 est produite par un promoteur alternatif P3 localisé dans TP53. Nous avons montré que Δ133p53 est un gène cible de p53, qui transactive le promoteur P3 par fixation sur un élément de réponse présent dans l’exon 4. L’expression de Δ133p53 est corrélée à celle d’autres gènes cibles de p53 en réponse à un stress génotoxique. De plus, elle réprime la suppression de la prolifération induite par p53 en inhibant ses capacités de liaison à l’ADN. Δ40p53 est produite par épissage alternatif, dont la rétention de l’intron 2 favorise sa traduction et empêche celle de p53. Nous avons montré que des structures de type G-quadruplexes présentes dans l’intron 3 régulent l’exclusion de l’intron 2. Ces structures comprennent le polymorphisme TP53PIN3 (duplication de 16pb), qui change leur localisation et affecte l’expression des ARNm codant p53 et Δ40p53. De plus, nous avons montré que ce polymorphisme est associé à une accélération de la cancérogenèse dans le syndrome Li-Fraumeni, caractérisé par la présence d’une mutation germinale TP53 (effet modificateur: 19 ans de différence à l’âge moyen du premier diagnostique entre les deux variants). L’expression des isoformes de p53 dépend de mécanismes transcriptionnels différents, indiquant des rôles différents dans la modulation des fonctions suppressives de p53. En plus d’inactiver p53 dans les cancers, ces isoformes pourraient être à l’origine des effets modificateurs des polymorphismes de TP53 sur les mutants p53. / The TP53 tumour suppressor gene expresses several isoforms, of which Δ40p53 (lack of transactivation domain) and Δ133p53 (lack of both transactivation and part of DNA-binding domains). These isoforms inhibit p53 suppressive activity and have been shown to be over-expressed in cancers (breat and melanoma). In cancers associated with low TP53 mutation rate, these isoforms could be great candidates to inactivate p53. It seems important to understand the transcriptional mechanisms that regulate their expression. Δ133p53 is produced by an alternative P3 promoter within TP53. We showed that Δ133p53 is a p53 target gene. p53 transactivates the P3 promoter and interact with a response element within exon 4. Δ133p53 expression is correlated to other p53 target genes in response to genotoxic stress. In addition, Δ133p53 inhibits p53-dependent suppression of proliferation by inhibiting p53 DNA-binding activity. Δ40p53 is produced by alternative splicing: retention of intron 2 favours its translation while it avoid the one of p53. We showed that G-quadruplex structures are formed in intron 3 and regulate retention of intron 2. The TP53PIN3 polymorphism (16 bp duplication) is embedded within these structures and affects their locations leading to variation of mRNA expression of p53 and Δ40p53. In addition, we showed that this polymorphism is associated with acceleration of carcinogenesis in Li-Fraumeni syndrome, characterized by germline TP53 mutation (genetic modifier effect: difference of 19 years in mean age at first diagnosis of cancer between the two variants). The expression of p53 isoforms depends on different transcriptional mechanisms, suggesting different roles in the modulation of p53 suppressive functions. In addition to inactivate p53 in cancers, these isoforms could be the mediators of modifier effects observed for TP53 polymorphisms on mutant p53.

O melanoma cutâneo é a neoplasia de pele de maior mortalidade. A imprevisibilidade de sua evolução é uma de suas características principais, o tratamento do tumor primário é, atualmente, de pouca morbidade e, na doença disseminada, as opções terapêuticas são pouco eficazes. É fundamental a pesquisa de marcadores tumorais que permitam a previsão da evolução, melhor compreensão da patogênese do melanoma e possibilitem a descoberta de alvos moleculares. Nesse contexto, estudos genéticos mostraram a importância da regulação do ciclo celular, especialmente a passagem da fase G1-S. Importantes fatores envolvidos compõem a cascata da proteína Rb (p16, ciclina D1, CDK4 e pRb) e da proteína p53 (p53 e p21). Objetivo: verificar a frequência da expressão de p16, ciclina D1,CDK4, pRb, p53 e p21 em melanomas cutâneos de cabeça, pescoço e tronco e sua relação com prognóstico. Métodos: Estudo retrospectivo envolvendo 46 pacientes (sendo 67,3% homens, idade média 57,7 ± 15,8 anos) com melanoma cutâneo de cabeça, pescoço e tronco que foram tratados pela mesma equipe com seguimento mínimo de dois anos. Foram estudados fatores clínicos (topografia do tumor primário, tempo de seguimento, ocorrência de metástases e óbito relacionado), histopatológicos (tipo histológico, índice de Clark, índice de Breslow) e análise imuno-histoquímica pela técnica de micro-array para as proteínas reguladoras do ciclo celular p16, ciclina D1, CDK4, pRb, p53 e p21. Resultados: Houve proporção igualitária entre as topografias (23 casos em tronco, 23 em cabeça e pescoço). Treze pacientes com Clark I (29,5%), cinco com II (11,3%), 16 com III (36,5%), 10 com IV (22,7%) e nenhum com Clark V. A média das medidas de Breslow foi 0,96 (DP=1,01). O seguimento médio foi de 77 meses (DP=47). Oito dos 46 pacientes (17,3%) tiveram evolução desfavorável, com seis óbitos relacionados. A idade foi mais elevada no grupo com evolução desfavorável (p=0,04). Houve expressão de p16 em 80%, ciclina D1 em 58,9%, CDK4 em 43,5%, pRb em 58,5%, p53 em 53,6% e p21 em 52,3% dos melanomas. Em análise univariada, a expressão do p21 foi relacionada com evolução desfavorável (p=0,04), o que não foi observado com a expressão dos outros marcadores (p>0,05). Conclusão: A expressão da proteína p21 nos melanomas cutâneos de cabeça, pescoço e tronco foi relacionada com evolução desfavorável, o que não ocorreu com outros fatores envolvidos na regulação do ciclo celular / Melanoma is the most lethal skin cancer. The outcome of melanoma is not predictable in most cases. Although the treatment for the primary tumor is well tolerated, there are no effective therapeutic options in disseminated disease. Efforts are being made in the search for tumoral markers that may predict outcome, increase the comprehension of melanoma pathogenesis, and may also help the search for molecular targets. In this issue, genetic studies concerning the regulation of cell cycle, including the G1-S checkpoint, are important. The retinoblastoma protein (pRb) pathway (p16, cyclin D1, CDK4 and pRb) and the p53 pathway (p53 and p21) are part of this regulation. Objectives: to verify the expression of p16, cyclin D1, CDK4, pRb, p53 and p21 in head, neck and trunk melanomas, and its correlation with prognosis. Method: Retrospective study approved by institution ethics committee. Fourtysix head, neck and trunk melanoma patients (67.3% men, mean age 57.7±15.8) treated by a single surgeon with minimum 2-years follow-up were enrolled. Clinical factors (primary tumor location, follow-up period, metastasis or related deaths), pathologic (histological subtype, Clark and Breslow index) and microarray immunohystochemical analysis of the cell cycle proteins p16, cyclin D1, CDK4, pRb, p53 and p21. Results: Location of the primary tumor was equal for head/neck and trunk (50% each). Thirteen patients were classified as Clark I (29.5%), five as Clark II (11.3%), 16 as Clark III (36.5%), 10 as IV (22.7%), none as Clark V. Mean Breslow measure was 0.96±1.01. Mean follow-up was 77±47 months. Eight patients (17.3%) had bad outcome, with six related deaths. Patients with worse outcome had a higher mean age at diagnosis (p=0,04). Expression of p16 was positive in 80%. Cyclin D1 was positive in 58.9%. CDK4 was positive in 43.5%. pRb was positive in 58.5%. p53 was positive in 53.6%. p21 was positive in 52.3%. Univariated analysis showed that p21 expression was related to worse outcome (p=0,04), while the other markers were not (p>0,05). Conclusion: p21 expression in head, neck and trunk melanomas was related to worse outcome. Expression of the other cell cycle regulators proteins was not

A síndrome de Li-Fraumeni (LFS) e sua variante like (LFL) são associadas a mutações germinativas no gene TP53 e predispõe ao alto risco para múltiplos tumores em idade jovem. Analisamos 91 famílias LFS/LFL do sul/sudeste do Brasil para mutações germinativas e haplótipos de TP53 (PIN2, PIN3 e PEX4) e MDM2 (309T-G). A mutação R337H ocorreu em 44,4% das famílias avaliadas. Em 750 controles da região a freqüência populacional da mutação foi 0,3%. A genotipagem de oito indivíduos não relacionados R337H-positivos para 29 TAG SNPs intragênicos demonstrou o mesmo haplótipo raro estabelecendo efeito fundador para R337H. O alelo duplicado no PIN3 apresenta impacto modificador e retardo de 17,1 anos na ocorrência de tumores em famílias com mutação no TP53, enquanto o SNP309 MDM2 modula a idade dos sarcomas de partes moles. / Li-Fraumeni syndrome (LFS) and its variant like (LFL) are associated with germline mutations in the TP53 gene and predispose to a variety of cancers at an earlier age. We analyzed 91 LFS/LFL families from southern Brazil for germline mutations in TP53 and polymorphisms in TP53 (PIN2, PIN3, PEX4) and MDM2 (309T-G). The germline TP53 mutation R337H was found in 44.4% of all families included. In 750 controls from the same region, mutation prevalence was 0.3%. Genotyping of eight unrelated R337H-positive individuals for 29 intragenic TAG SNPs showed that they all shared the same rare haplotype confirming the founder effect for the mutation. Duplication of PIN3 had a modifier effect on the age of tumor onset (delay of 17.1 years) in TP53 mutation carriers whereas MDM2 SNP309 modulated age of onset for soft-tissue sarcomas.

As proteínas p53, MDM-2, c-Kit, ErbB-2, PCNA e p-glicoproteína foram estudadas em 42 amostras de osteossarcoma ao diagnóstico, através da técnica de imunohistoquímica, e foram correlacionados estes achados com o prognóstico destes pacientes. O p-53 foi positivo em 23,1% (9/39), PCNA em 71,4% (25/35), p-glicoproteína em 40,5% (15/37), MDM-2 em 34,8% (8/23), c-kit em 67,6% (25/37) e ErbB-2 em 17,9% (7/39). Na análise univariada, a presença de metástases ao diagnóstico, a positividade de p53 e ErbB-2 influenciaram o prognóstico individualmente, entretanto, na análise multivariada, a presença de metástase ao diagnóstico revelou-se o único fator de prognóstico estatisticamente significante / We study, by imunohistochemistry technique, the proteins p53, MDM-2, c-Kit, ErbB-2, PCNA and p-glycoprotein in samples of osteosarcoma tumors at diagnosis and its correlation with the prognosis of this patients. The p-53 was positive in 23,1% (9/39), PCNA in 71,4% (25/35), p-glycoprotein in 40,5% (15/37), MDM-2 in 34,8% (8/23), c-kit in 67,8%(25/37) and ErbB-2 in 17,9% (7/39) of the samples. In the univariate analysis, the presence of metastasis at diagnosis, the positivity of p-53 and ErbB-2 influenced the prognosis individually, otherwise, in the multivariate analysis, the presence of metastasis at diagnosis was the only prgnostic factor statistically significant