Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 171 pages. Includes vita. Includes bibliographical references.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 124-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Biochemistry) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 69-83). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Orientadores: Sophie Fraçoise Mauricette Derchain, Luís Otávio Zanatta Sarian / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T00:11:32Z (GMT). No. of bitstreams: 1 Serra_KatiaPiton_D.pdf: 3794654 bytes, checksum: ce5565714883e2e12d99e6f9ed0ca141 (MD5) Previous issue date: 2014 / Resumo: Introdução: Na última década, doferentes subtipos moleculares de cancer de mama foram propostos. A classificação clinic-patológicas dos subtipos vem comprovando ser estratégica para predizer sobrevida e resposta ao tratamento. Modificação recente da classificação considera a avaliação semiquantitativa da expressão dos RP no curso clínico e resposta ao tratamento. Embora exista associação apreciável com o prognóstico e indicação de terapia citotóxica e endócrina, os subtipos parecem falhar em explicar completamente o comçortamento da doença e a resposta ao tratamento. Moléculas como as da família das cicloxigenases (COX), composta por três entidades (COX 1, 2 e 3) vem demonstrando associação com a carcinogênese mamária, e a análise da expressão da p53 nos tumores de mama pode também oferecer informações adicionais para determinação do prognóstico. Objetivos: Foi avaliada a associação entre os subtipos clinic-patológicos do cancer de mama com o prognóstico e fatores preditivos em uma relativamente grande casuística de pacientes Brasileiras com câncer de mama, que foram acompanhadas por cerca de quatro anos. Foram discutidas as vantagens e possíveis ressalvas relacionadas à nova classificação. Também foi mensurada a expressão da COX2 e da p53 em relação aos subtipos clínico-patológicos e avaliada se a expressão destas molécular poderia explicar a variabilidade no prognóstico ainda encontrada entre os subtipos clínico-patológicos do câncer de mama. Metodologia: Um total de 183 amostras de cancer de mama foram obtidas de mulheres tratadas no Hospital da Mulher da Universidade Estadual de Campinas, Campinas, Brasil, entre Junho de 2008 e Janeiro de 2011. Tissue microarrays (TMA) foram construídos dos blocos originais de parafina para realização de imunoistoquímica (IQ) e hibridização fluorescente in situ (FISH). IQ foi realizada para detecção da expressão de RE, RP, ki67, COX2 e p53; o status do HER2 foi avaliado por FISH nas 183 amostras. Os tumores foram classificados em cinco categorias de acordo com a definição correspondente clinic-patológica dos dos subtipos intrínsecos do câncer de mama, definida durante a 13th St Gallen International Breast Cancer Conference (2013). As características clínicas e patológicas das pacientes e seus tumors e a sobrevida foi avaliada em relação aos subtipos clínico-patológicos, a COX2 e a p53. O tempo médio de seguimento foi 2,94 anos (90% faixa central = 0,93 a 4,1 anos). Resultados: Aproximadamente 75% dos tumors foram classificados como luminais-like. OS HER2 positivos (não luminais) somaram 9,3% dos casos e os Triplos-negativos 13,1%. Os Luminais B-like e HER2 positivos (não luminais) foram associados a alto grau histológico quando comparados aos Luminais A-like (p<0,01). Os Luminais A-like associaram-se significativamente com melhor sobrevida global e livre de doença quando comparados aos HER2 positivos (não luminais) e Triplos-negativos. Não houve tendência à expressão de COX2 relacionada aos subtipos de Luminal A-like a Triplo-negativo. Em contraste, a p53 se expressou em cerca de 67% dos tumores Luminais A-like, 50% dos Luminais B-like HER2 positivos, 60,9% dos Luminais B-like HER2 negativos, 82% dos HER2 positivos (não luminais) e 87% dos Triplos-negativos (p para tendências = 0.06). Houve uma significativa expressão de COX2 nos tumors (66,9%) quando a p53 eram também positive, comparada àqueles tumors que não expressavam p53 (em cujo caso apenas 18,0% dos tumores foram positivos para COX2; p<0,001). Nem a COX2, nem a p53 se relacionaram à sobrevida das pacientes. Conclusões: O critério mais estrito para definer os tumors Luminais A-like aumentou a acurácia da classificação para selecionar tumors que partilhem um bom prognóstico e respondam a terapia endócrina. Parece haver uma associação positive entre a expressão da COX2 e da p53. Por outro lado, nem a expressão da COX2 nem a da p53 se associaram aos subtipos clínico-patológicos, características clínicas e do tumor e ao prognóstico. Parece ser muito cedo para eleger a detecção de COX2 usando IQ como ferramenta de prognóstico ou preditiva, mas evidências incipientes apontam para um possível papel para o marcador / Abstract: Background: In the last decade, different molecular subtypes of breast cancer have been proposed. The clinico-pathological surrogate subtypes of breast cancer classification has been proven as straightforward strategy to predict patient survival and response to treatment. Recent modifications to the classification considered the semi quantitative evaluation of the expression of PR in the clinical course and response to treatment. Although displaying appreciable association with disease prognosis and the prognostic value of cytotoxic and endocrine therapeutic modalities, the subtypes seem to fail at completely explaining disease behavior and response to treatment. Molecules such as those of the cyclocooxigenase (COX) family, currently composed of three entities (COX 1, 2 and 3) have been shown to be associated with breast carcinogenesis, and the analysis of p53 expression in breast tumors may also offer some additional prognostic clues. Objectives: We tested the association of the current clinico-pathological surrogate subtypes of breast cancer with the main prognostic and predictive factors in a relatively large dataset of breast cancer Brazilian patients, which were followed up for almost four years. We discuss the advantages and possible caveats related to this new classification. Our study also assessed COX2 and p53 expression in these clinico-pathological subtypes, and evaluated whether the expression of these molecules could help further explain the variability in prognosis still found within the surrogate molecular groups of breast cancer. Methods: A total of 183 breast cancer samples were obtained from women treated at the Women's Hospital of Campinas State University, Campinas, Brazil, between June 2008 and January 2011. Tissue microarrays (TMA) were constructed from the original paraffin blocks for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analyses. Immunohistochemistry was performed to detect the expression of ER, PR, ki67, COX2, and p53; the HER2 status of the 183 specimens was assessed using FISH. Tumors were subtyped into five distinct categories according to the Clinico-Pathological surrogate definitions of intrinsic subtypes of breast cancer defined during the 13th St Gallen International Breast Cancer Conference (2013). Clinical and pathological features of patients and their tumors, and patients¿ survival were assessed in relation to the surrogate subtypes, COX2 and p53. Mean follow-up time was 2.94 years (90% central range = 0.93 to 4.1 years). Results: Approximately 75% of the tumors were classified as luminal-type-like. HER2 positive (non-luminal) tumors accounted for 9.3% of the cases and Triple-negative tumors for the remainder 13.1%. Luminal B-like and HER2 positive (non-luminal) tumors were associated with higher histological grades when compared to Luminal A-like tumors (p<0.01). Luminal A-like tumors were significantly associated with better disease free and overall survival when compared to HER2 positive (non-luminal) and Triple-negative tumors. There was no trend in COX2 overexpression from Luminal A to Triple-negative subtypes. By contrast, p53 was expressed in roughly 67% of the Luminal A-like tumors, 50% of the Luminal B-like HER2 positive tumors, 60.9% of the Luminal B-like HER2 negative, approximately 82% of the HER2 positive (non-luminal) and 87% of the Triple-negative tumors (p for trends = 0.06). There was a significantly higher proportion of COX2 positive tumors (66.9%) when p53 was also positive compared to when the tumor was negative for p53 (in which case only 18.0% of the tumors were positive for COX2; p<0.001). Neither COX2 nor p53 were found to be associated with patients¿ survival. Conclusions: The more strict criteria to define Luminal A-like tumors increased the accuracy of the classification by selecting tumors that share a good prognosis and response to endocrine therapy.There seems to be a positive association between the expressions of COX2 and p53. On the other hand, neither the expression of COX nor that of p53 was associated with clinic-pathological subtypes, tumor features and prognosis. It seems to be too early to elect the detection of COX2 using IHC as prognostic or predictive tool, but incipient evidence points towards a possible role for the marker / Doutorado / Oncologia Ginecológica e Mamária / Doutora em Ciências da Saúde

ARF, a potent tumor suppressor, positively regulates p53 by antagonizing MDM2, a negative regulator of p53, which in turn, results in either apoptosis or cell cycle arrest. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF-null or p53-null mice, results in a broadened tumor spectrum and decreased tumor latency. This evidence suggests that ARF exerts both p53-dependent and p53-independent tumor suppressor activity. However, the molecular pathway and mechanism of ARF’s p53-independent tumor suppressor activity is not understood. The antiapoptotic, metabolically regulated, transcriptional corepressor C-terminal binding protein 2 (CtBP2) has been identified as a specific target of ARF’s p53-independent tumor suppression. CtBPs are phosphoproteins with PLDLS-binding motif and NADH-binding central dehydrogenase domains. ARF interacts with CtBP1 and CtBP2 both in vitro and in vivo, and induces their proteasome-mediated degradation, resulting in p53-independent apoptosis in colon cancer cells. ARF’s ability to target CtBP2 for degradation, and its induction of p53-independent apoptosis requires an intact interaction with CtBP2, and phosphorylation at S428 of CtBP2. As targets for inhibition by ARF, CtBPs are candidate oncogenes, and their expression is elevated in a majority of human colorectal adenocarcinomas specimens in comparison to normal adjacent tissue. Relevant to its targeting by ARF, there is an inverse correlation between ARF and CtBP expression, and CtBP2 is completely absent in a subset of colorectal adenocarcinomas that retains high levels of ARF protein. CtBPs are activated under conditions of metabolic stress, such as hypoxia, and they repress epithelial and proapoptotic genes. BH3-only genes such as Bik, Bim and Bmf have been identified as mediators of ARF-induced, CtBP2-mediated p53-indpendent apoptosis. CtBP2 repressed BH3-only genes in a tissue specific manner through BKLF (Basic kruppel like factor)-binding elements. ARF regulation of BH3-only genes also required intact interaction with CtBP2. ARF antagonism of CtBP repression of Bik and other BH3-only genes may play a critical role in ARF-induced p53-independent apoptosis, and in turn, tumor suppression. To study the physiologic effect of ARF/CtBP2 interaction at the organismal level, the p19ArfL46D knock-in mice, in which the Arf/CtBP2 interaction was abrogated, was generated. Analysis of the primary cells derived from these mice, revealed that the Arf/CtBP2 interaction contributes to regulation of cell growth and cell migration. Overexpression of CtBP in human tumors, and ARF antagonism of CtBP repression of BH3-only gene expression and CtBP-mediated cell migration may therefore play a critical role in the p53-independent tumor suppressor function/s of ARF.

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.

<p>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.</p><p>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 <i>Cyp2a5/CYP2A6</i>. 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 <i>cis</i> 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. </p><p>The data presented here further suggest that the transcriptional and post-transcriptional processes controlling the expression of the murine <i>Cyp2a5</i> gene are linked <i>via</i> hnRNP A1, by performing functions in the nucleus as a transcription factor, or in the cytoplasmic compartment as a <i>trans </i>factor bound to the 3’UTR of the mRNA as needed. Our studies of the human ortholog of this gene, <i>CYP2A6</i>, suggest that this gene is regulated post-transcriptionally in a manner similar to that of its murine counterpart, <i>via</i> changes in mRNA stability and interaction of hnRNP A1 with its 3’ UTR. </p>

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 131 pages. Includes vita. Includes bibliographical references.