By Western blotting and immunohistochemical analyses, we have demonstrated that Pin1 was overexpressed in 71.4% of hepatocellular carcinoma (HCC) and its levels correlated with the clinical survival rate. This conclusion was supported by the results from examining Pin1 protein in HCC cancer cell lines. RT-PCR was performed to examine the Pin1 transcription level in tumor part and was compared with that in non-tumor part. Our results indicated that pin1 overexpression was due to the upregulation of Pin1 transcription. Interestingly, most of the cases with upregulation of Pin1 have been shown to correlate with £]-catenin and Cyclin D1 accumulation in HCC specimens. These results were consistent with the previous studies that Pin1 caused £]-catenin and Cyclin D1 elevation in breast cancer. The concordance between hepatitis virus chronic infection and Pin1 overexpression of HCC patients was also analysis. Taken together, these data indicated that Pin1 overexpression leading to £]-catenin and Cyclin D1 accumulation might play a critical role in hepatocellular carcinogenesis and tumor progression. Pin1 levels therefore can be used as a prognostic marker for HCC, and our results suggested that Pin1 is a potential target for therapeutic intervention in hepatocellular carcinoma.

ABSTRACT Chibby (or PIGEA-14) is a novel antagonist of the Beta-catenin pathway in nucleus. However, the tumor-suppressing function of Chibby and the importance of nuclear targeting to the cellular functions of Chibby have not been validated. By fusion of Chibby cDNA with green fluorescent protein (GFP) or Flag-tag, it was found that exogenous Chibby expression was detected in the nucleus as well as cytoplasm of transfected HeLa cells, but with a preferential nuclear localization (more than 50% cells with nuclear Chibby expression). Chibby overexpression significantly abrogated the cellular Beta¡Vcatenin activities and induced apoptosis in HeLa cells. Moreover, Chibby gene delivery attenuated the proliferation, migration, and anchorage-independent growth of HeLa cells, supporting the tumor suppressor function of Chibby. Mutation or deletion of the predicted nuclear localization sequence (NLS), at residues 123-126, significantly promoted the cytoplasmic localization of Chibby, indicating residues 123-126 is the NLS domain of Chibby. Interestingly, ecotopic expression of Chibby NLS mutants remained capable of inducing apoptosis and inhibiting Beta¡Vcatenin activities in HeLa cells. Besides, overexpression Chibby NLS mutants effectively attenuated the viability, motility and colonies formation of HeLa cells. Expression analysis revealed that Chibby NLS mutants retained Beta-catenin in the cytoplasm and prevented its nuclear entry, thereby inhibiting the Beta-catenin transcriptional activities. In summary, Chibby shuttles between nucleus and cytoplasm, and possesses the functions of tumor suppressor and Beta-catenin antagonist.

The epidermal growth factor family of receptors is important in the development and progression of many types of cancers including, breast, lung, and glioblastoma. The family consists of 4 members (EGFR/erbB1, Her2/erbB2, erbB3, and erbB4). In all breast cancer cases, EGFR expression is deregulated 20 to 30% of the time; however in the most aggressive form of breast cancer (basal-like) EGFR expression is upregulated in 60% of cases. EGFR's expression and activity can be altered in transformed cells through a variety of mechanisms, such as novel protein-protein interactions, gene amplification, mutations, and loss of regulatory proteins. In this work we have examined the role of cancer specific protein interactions of EGFR with MUC1 and beta-catenin in the progression of breast cancer.Herein I report that the interaction of MUC1 and EGFR in breast cancer cells alters EGFR localization by promoting EGFR nuclear translocation. Importantly, I discovered that the presence of MUC1 mediates EGFR's interaction with chromatin. More specifically, I found that EGFR interacts with the cyclin D1 promoter region in a MUC1-dependent fashion which resulted in a significant increase in cyclin D1 protein expression. Nuclear EGFR localization has been shown to correlate with resistance to anti-EGFR therapies, which indicates that MUC1's interaction with EGFR could be a mechanism of resistance.MUC1's interaction with both EGFR and beta-catenin can promote transformation therefore a peptide therapy was developed, PMIP, which mimics the hypothesized interaction domains of MUC1's cytoplasmic tail. PMIP was designed to inhibit the interaction of MUC1/EGFR and MUC1/beta-catenin thereby regulating EGFR expression and promoting beta-catenin localization to adherens junctions. PMIP effectively enters the cytosol of cells and inhibits the target interactions. Importantly, PMIP inhibited invasion and proliferation of breast cancer cells and in mice significantly reduced the growth rate of breast cancer xenograft and genetically-driven tumors. This study demonstrated that the use of peptides to inhibit intracellular protein interactions is a viable option that would have limited toxic side-effects. Overall, this work reveals a new regulatory role of EGFR localization and activity by MUC1 and that this mechanism is viable therapeutic breast cancer target.Lastly, in a mouse model of breast cancer I examined the role of EGFR tyrosine kinase activity in beta-catenin dependent tumorigenesis. A transgenic mouse model of breast cancer, MMTV-Wnt-1, was bred onto an EGFR kinase deficient background. I discovered that the loss of EGFR kinase activity in this model resulted in a significant delay in tumor onset and inhibited tumor growth. These findings indicate a cooperation of EGFR and beta-catenin dependent signaling pathways, which promote transformation of glandular epithelial cells.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 133-173).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 44-52).

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.

PART1, TITLE: A p53-inducible long non-coding RNA PICART1 mediating cancer cell proliferation and migration. Long non-coding RNAs (lncRNAs) function in the development and progression of cancer, but only a small portion of lncRNAs are characterized thus far. A novel lncRNA transcript with 2.53 kb in length was identified by a transcriptome sequencing analysis, named p53-inducible cancer-associated RNA transcript 1 (PICART1). This PICART1 is upregulated by p53 through a p53-binding site at -1808 to -1783bp. In breast and colorectal cancer cells and tissues, PICART1 expression was decreased. Ectopic expression of the PICART1 suppressed growth, proliferation, migration, and invasion of MCF7, MDA-MB-231 and HCT116 cells whereas silencing of PICART1 stimulated the cell growth and migration. In these cells, the expression of PICART1 lowered down the levels of p-AKT (Thr308 & Ser473) and p-GSK3β (Ser9), and accordingly, β-catenin, cyclin D1 and c-Myc expression were decreased, but p21cip1/Waf1 expression was increased. Together these data suggest that PICART1 is a novel p53-inducible tumor suppressor lncRNA, functioning through the AKT/GSK3β/β-catenin signaling cascade. PART2, TITLE: The novel long non-coding RNA PANCR is a tumor suppressor gene in breast cancer. Long non-coding RNAs (lncRNAs) function as oncogenes or tumor suppressors in development and progression of cancer. Chromosome 16q22.1 region is frequently deleted in breast cancer, which may contribute to breast carcinogenesis by inactivation of tumor suppressor genes. This study characterized a new lncRNA tumor suppressor, named p53 activating non-coding RNA (PANCR), located in this Chromosome 16q22.1 region. This PANCR lncRNA consists of 1.5kb in length. Our data showed that PANCR was downregulated in breast cancer tissues and cell lines. In the breast cancer cell lines, PANCR expression appeared reversely correlated with cell malignancy, and in breast cancer tissues, PANCR was downregulated over 2 times in 31 (62.0%) of 50 cases when compared to adjacent normal breast tissues. In breast cancer cells MCF7 cells, ectopic expression of PANCR suppressed cell proliferation in culture, but in contrast, shRNA–mediated silencing of PANCR promoted cell growth and proliferation.

Orientador: Antonio Gonçalves de Oliveira Filho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T04:54:32Z (GMT). No. of bitstreams: 1 Patricio_FlaviaRezendePereira_M.pdf: 2174141 bytes, checksum: 0011ea137b3f271896d984e54bb4f4d6 (MD5) Previous issue date: 2009 / Resumo: Tumores do córtex da supra-renal (TCSR) em crianças são raros e correspondem a 0,2% dos tumores da infância. Estes tumores são endocrinologicamente ativos, causando na maioria das vezes, virilização do paciente associada, ou não, a um aumento de cortisol. O tratamento dos TCSR é principalmente cirúrgico, sendo a cirurgia com o procedimento de ressecção completa do tumor e sem ruptura a principal modalidade terapêutica. No entanto, a distinção entre tumores benignos e malignos, baseada exclusivamente na histologia, pode ser difícil de ser realizada. Os fatores prognósticos são baseados quase exclusivamente no estadiamento da doença, o qual leva em conta o peso e volume tumoral e a disseminação metastática do tumor. Estudos clínicos e experimentais sugerem que a propagação metastática em alguns tumores está relacionada aos níveis de E-caderina e ?-catenina, que são moléculas presentes no tecido epitelial normal, estando envolvidas diretamente na adesão intercelular. A análise da expressão destas moléculas pode fornecer dados que permite identificar grupos de pacientes mais propensos à evolução tumoral desfavorável, proporcionando, assim, um tratamento mais adequado e individualizado a estes pacientes. Com o objetivo de analisar a expressão da E-caderina e ?-catenina em crianças com TCSR e sua correlação com a evolução da doença, foi realizada uma revisão retrospectiva dos prontuários de 33 crianças com diagnóstico de TCSR tratadas no Centro Infantil Boldrini (Janeiro de 1998 a Janeiro de 2005). Foram coletados e analisados dados referentes ao sexo, idade, manifestações clínicas, estadiamento, tratamento e evolução dos pacientes. Para a análise imunoistoquímica, foi empregada a técnica de multitissue array com anticorpos específicos para E-caderina e ?-catenina em 30 tumores de crianças com TCSR e uma adrenal normal. Houve predominância do sexo feminino na amostra e a apresentação clínica mais freqüente foi a virilização. Nesta série, observou-se que crianças com idade inferior a dois anos apresentaram melhor prognóstico e, também que a ruptura e recidiva tumoral apresentaram influência negativa na sobrevida dos pacientes. A análise imunoistoquímica mostrou expressão da E-caderina em 73,3% e ?-catenina em 83,3% das crianças que apresentavam TCSR. Além disso, não foi verificada sua expressão na glândula adrenal normal. Quando avaliada a relação da expressão da E-caderina e ?-catenina com os estádios evolutivos da doença, não foi verificada associação significativa entre as variáveis. A positividade da E-caderina e ?-catenina na membrana celular, citoplasma ou núcleo, verificou-se que a expressão na membrana celular mostrou associação significativa com mau prognóstico / Abstract: Adrenal cortical tumor (ACT) in children are rare and they correspond to 0,2% of the tumors of the childhood. They are usually active, causing mainly virilization of patient associate or not with increased levels of corticoids. The treatment of the TCSR is mainly surgical, being the surgery with complete resection of the tumor without spillage the therapeutic mainstay. The distinction between benign and malignant tumors based exclusively on the histology can be difficult and very often uncertain. The prognostic factors are based almost exclusively on the staging of the disease, who takes into account the weight and volume tumoral and the metastatic dissemination. Clinical and experimental studies suggest that metastatic dissemination in some tumors is related with the levels of E-cadherin and ?-catenin. These molecules are found in the normal epithelial tissues and are strongly related with intercellular adhesion. The analysis of the expression of these molecules maybe can allow identifying groups of patients with higher risk of presenting unfavorable outcomes and ensuing appropriate and individualized treatment. With the objective of analyzing the expression of E-cadherin and ?-catenin in children with ACT and its correlation with the evolution of the disease, a retrospective chart review of 33 children with diagnosis of ACT treated at Centro Infantil Boldrini (January of 1998 through January of 2005). Data regarding sex, age, clinical presentation, staging, treatment and outcome were collected and analyzed. Multitissue array technique using specific antibodies for E-cadherin and ?-catenin was done in 30 tumors from children with ACT and 1 normal adrenal tissue. There was predominance of the feminine sex and the most frequent clinical presentation was virilization. In these series children with age bellow two years had a better outcome and tumoral spillage and relapse have had negative influence in the survival of the patients. Immunohistochemical analysis showed expression of E-cadherin in 73,3 % and ?-catenin in 83,3 % of the children who had ACT but showed no expression in the normal adrenal tissue. When the relationship between the expression of E-cadherin and ?-catenin with the stages of the disease was analyzed, no significant association was found. When analyzed the expression of E-cadherin and ?-catenin in the cellular membrane, cytoplasm or nucleus, its presence in the membrane of the cell was found as associated with poor outcome. As far we know, this is the first study to evaluate the expression of E-cadherin and ?-catenin in children with ACT and, although with small number of patients due to the rarity of the disease, it apparently shows some relationship with prognosis. When the relationship between the expression of E-cadherin and ?-catenin with the stages of the disease was analyzed, no significant association was found. When analyzed the expression of E-cadherin and ?-catenin in the cellular membrane, cytoplasm or nucleus, its presence in the membrane of the cell was found as associated with poor outcome. As far we know, this is the first study to evaluate the expression of E-cadherin and ?-catenin in children with ACT and, although with small number of patients due to the rarity of the disease, it apparently shows some relationship with prognosis / Mestrado / Cirurgia / Mestre em Cirurgia

Die Regulation des Wnt/β-catenin Signalwegs ist nicht nur entscheidend für alle Stadien der kardialen Entwicklung, sondern auch für die Homöostase im adulten Herzen. Tatsächlich ist die Aktivierung des Wnt/β-catenin Signalwegs mit dem pathologischen Herz-Remodeling assoziiert. Ein besseres Verständnis der Regulation des Wnt/β-catenin Signalwegs bei Herzinsuffizienz könnte die Identifikation potentieller Faktoren zum Blockieren des pathologischen Herz-Remodelings und/oder das Aktivieren der endogenen Regeneration ermöglichen. Diese Arbeit konzentriert sich auf die Identifikation von gewebespezifischen Regulatoren des Wnt/β-catenin Signalwegs im Herzen. Frühere Arbeiten unserer Gruppe identifizierten basic leucine zipper and W2 domains containing protein (BZW) 2 als einen kardialen Interaktionspartner von β-catenin und KLF15. Die Rolle von BZW2 im Wnt/β-catenin Signalweg bei der Kardiogenese und der Homöostase des adulten Herzens war das Thema dieser Studie. Eine Analyse des BZW2-Proteins zeigte, dass die mutmaßliche ZIP und bZIP umfassende Domäne wichtig für die nukleare Lokalisation ist. Die Expression von BZW2 hat sich als bedeutsam im adulten Herzen, aber auch während der embryonalen Kardiogenese erwiesen. Eine niedrige BZW2-Expression ist für die effektive Bildung des kardialen Mesoderms notwendig, denn es kam unter BZW2-Überexpression zu einem Abbruch der Kardiomyozytenbildung in einem Modell der in vitro Kardiogenese. Dennoch war die BZW2-Expression nicht entscheidend für die Embryogenese, was auf eine kompensatorische Funktion verwandter Proteine hindeuten könnte. Interessanterweise war BZW2 für den Erhalt der normalen Herzfunktion und während der Reaktion auf Stress notwendig. Obwohl BZW2 die Wnt-Transkriptionsaktivität in vitro nicht signifikant inhibiert hat, resultierte das Fehlen von BZW2 in de novo Synthese von β-catenin spezifisch im adulten Herzgewebe in vivo. Außerdem wurde BZW2 selbst durch den Wnt/β-catenin Signalweg reguliert, was auf seine Rolle als regulatorischer Rückkopplungsfaktor in vivo hindeutet. Zusammenfassend identifizierte diese Arbeit einen neuen herzgewebespezifischen Faktor des Wnt/β-catenin Signalwegs auf einer neuen Regulationsebene und demonstrierte seine Relevanz für die normale Herzhomöostase. Angesichts der ubiquitären Expression und der vielfältigen Funktionen von β-catenin könnte die gewebespezifische Modulation neue Therapieoptionen darstellen.

PROBLEM: Colorectal cancer (CRC) is a top three leading cause of death among western countries. Epidemiological evidence shows a positive correlation between western diet, which consists of high-fat, meat and processed foods. Positive correlations indicate that diets high in fruits and vegetables could greatly decrease risk of CRC. Specifically the ubiquitous spice, turmeric, and its main active constituent have been broadly researched to determine its efficacy in the treatment and prevention of CRC. RESULTS: Curcumin proves to be effective in the treatment and prevention of CRC. It acts as a chemosensitizer for chemotherapeutics which increases their effectiveness especially against chemoresistant CRC cell lines. In many in vitro studies curcumin has inhibited critical pathways involved in CRC progression such as Wnt/β-catenin and sonic hedgehog pathway. Curcumin can also act as a ligand for VDR, which is significant because high vitamin D intake is associated with a decreased risk of CRC. In vivo, curcumin has minimized tumor growth in animal models. In clinical trials curcumin proves to be a naturally derived, non-toxic agent. CONCLUSION: Curcumin and turmeric should be further studied for its use against CRC, specifically its use in nanotechnology and NDDS as either a stand-alone nutraceutical or a chemosensitizer. Additionally, it would likely be advantageous to prescribe turmeric in the diet in combination with black pepper, heat, and oil (which increases its bioavailability) in patients at high risk of developing CRC.