Orientador: Jose Barreto Campello Carvalheira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T10:00:11Z (GMT). No. of bitstreams: 1 Oliveira_JosenilsonCamposde_D.pdf: 1237045 bytes, checksum: 1e4b99f788f9635f40a69249e72e33ba (MD5) Previous issue date: 2009 / Resumo: A regulação adequada da via de sinalização PI 3-quinase-Akt é essencial para a prevenção da carcinogênese. Dados recentes caracterizaram uma alça de retroalimentação negativa na qual a mammalian target of rapamycin (mTOR), bloqueia a ativação adicional da via Akt/mTOR por meio da inibição da função do substrato 1 do receptor de insulina (IRS-1). Entretanto, a inibição potencial do IRS-1 durante o tratamento com rapamicina não foi estudado. No presente estudo, demonstramos que um oligonucleotídeo anti-sense direcionado ao IRS-1 e a rapamicina antagonizam sinergicamente a ativação da mTOR in vivo e induzem supressão tumoral, por meio de inibição da proliferação e indução de apoptose, em enxertos de células de câncer de próstata. Estes dados demonstram que a inclusão de agentes que bloqueiam o IRS-1 potencializam o efeito da inibição da mTOR no crescimento de enxertos de células de câncer de próstata. / Abstract: Proper activation of phosphoinositide 3-kinase-Akt pathway is critical for the prevention of tumorigenesis. Recent data have characterized a negative feedback loop where in mammalian target of rapamycin (mTOR), blocks additional activation of the Akt/mTOR pathway through inhibition insulin receptor substrate 1 (IRS-1) function. However, the potential of IRS-1 inhibition during rapamycin treatment has not been examined. Herein, we show that IRS-1 antisense oligonucleotide and rapamycin synergistically antagonize the activation of mTOR in vivo and induced tumor suppression, through inhibition of proliferation and induction of apoptosis, in prostate cancer cell xenografts. These data demonstrate that the addition of agents that blocks IRS-1 potentiate the effect of mTOR inhibition in the growth of prostate cancer cell xenografts. / Doutorado / Clinica Medica / Doutor em Clínica Médica

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

The activated product of the c-MYC proto-oncogene is one of the strongest known activators of carcinogenesis. It has been estimated that as many as one-seventh of all cancer deaths are associated with alterations in the c-MYC gene or its expression [1]. Therefore, understanding the regulation of c-MYC expression is a key factor in understanding carcinogenesis in many histologic classes of malignancy. The nuclease hypersensitive element (NHE) III₁ region of the c-MYC promoter has been shown to be particularly important in regulating c-MYC expression. Specifically, the formation of a G-quadruplex structure appears to promote repression of c-MYC transcription. In this dissertation, we investigate the role that nucleolin, a critical player in ribosome biogenesis and cell stress sensing, plays on the transcriptional regulation of the c-MYC promoter through its interaction with the c-MYC G-quadruplex structure. Our studies initiated with the design of a c-MYC G-quadruplex affinity column intended to trap potential c-MYC G-quadruplex-binding proteins that were then identified by LC-MS/MS. After careful examination of the literature of the list of potential c-MYC G-quadruplexbinding proteins, we realized that several of the proteins identified had been previously reported to interact directly with nucleolin. Consequently, we chose to focus our studies on nucleolin, as it could be a central regulator of the (NHE) III region. By performing chromatin immunoprecipitation in HeLa cells, we found that nucleolin indeed interacts with the c-MYC promoter region containing the NHE III₁ element. This binding activity was confirmed by both electromobility shift assay and polymerase stop assay. We provide evidence that nucleolin can induce the formation of the c-MYC G-quadruplex structure from single-stranded DNA, both in linear and circular DNA forms. We show that upon binding, nucleolin increases the stability of the c-MYC G-quadruplex structure leading to repression of c-MYC promoter activity. We also show that nucleolin binds with much higher affinity to G-quadruplex structures with topology similar to that of the parallel c-MYC G-quadruplex, such as those found in the VEGF and PDGF-A promoters; in comparison to G-quadruplexes found in telomeres or the c-MYB promoter, whose have significantly different topology. Interestingly, we also demonstrate that nucleolin binds with higher affinity to the c-MYC G-quadruplex than to its consensus RNA substrate, the nucleolin recognition element (NRE). Furthermore, we show that the C-terminal domain of nucleolin is critical for its interaction and stabilization of the c-MYC G-quadruplex structure. Lastly, we show that the binding of nucleolin to the (NHE) III region causes repression of c-MYC transcription. On the basis of these results, we propose that nucleolin may play an important role in the transcriptional regulation of c-MYC in vivo by inducing the formation of the c-MYC G-quadruplex structure.

Tang Wai-man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 142-147). / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.v / Lists of Abbreviations --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Isolation of Mas Oncogene --- p.1 / Chapter 1.2 --- Distribution of Mas Oncogene..........…… --- p.3 / Chapter 1.3 --- Developmental Expression of Mas Oncogene --- p.5 / Chapter 1.4 --- Study of Mas-deficient Mice --- p.7 / Chapter 1.5 --- Signal Transduction of Mas Oncogene --- p.8 / Chapter 1.6 --- Other Family Member of Mas Oncogene --- p.9 / Chapter 1.7 --- Mas and Angiotensin Receptor --- p.11 / Chapter 1.8 --- Angiotensin Receptors / Chapter 1.8.1 --- Classification of Angiotensin AT1 Receptor --- p.14 / Chapter 1.8.2 --- Cloning of Angiotensin Receptor --- p.15 / Chapter 1.9 --- Expression of Angiotensin Receptor / Chapter 1.9.1 --- Physiological Factors --- p.17 / Chapter 1.9.2 --- Cis-regulatory Elements / Chapter 1.9.2.1 --- Organization and Regulatory Elements of AT1 Receptor --- p.19 / Chapter 1.9.2.2 --- Expression of AT1a Receptor Promoter was Induced by AP-1 and GATA-4 in Pressure Overload Model --- p.20 / Chapter 1.9.2.3 --- AT1a Receptor Reveals Three Glucocorticoid Responsive Elements --- p.22 / Chapter 1.10 --- Signal Transduction of Angiotensin Receptor --- p.22 / Chapter 1.11 --- Aim of Project --- p.25 / Chapter Chapter 2: --- Mas Oncogene in AR4-2J cells / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Materials / Chapter 2.2.1.1 --- Reagents --- p.27 / Chapter 2.2.1.2 --- Enzymes --- p.27 / Chapter 2.2.1.3 --- DNA Purification Kits --- p.28 / Chapter 2.2.1.4 --- Materials and Antibodies for Western Blot --- p.28 / Chapter 2.2.1.5 --- Others --- p.28 / Chapter 2.2.2 --- Restriction Enzyme Digestion --- p.29 / Chapter 2.2.3 --- Agarose Gel Electrophoresis --- p.29 / Chapter 2.2.4 --- DNA Extraction and Purification --- p.29 / Chapter 2.2.5 --- Plasmid Vector Modification and DNA Ligation --- p.30 / Chapter 2.2.6 --- Bacterial Transformation --- p.31 / Chapter 2.2.7 --- Preparation of Plasmid DNA / Chapter 2.2.7.1 --- Minipreps --- p.32 / Chapter 2.2.7.2 --- Midipreps and Maxipreps --- p.33 / Chapter 2.2.8 --- Genomic DNA Extraction From Tissue and Cell Culture --- p.34 / Chapter 2.2.9 --- RT-PCR Cloning of Mas Oncogene --- p.35 / Chapter 2.2.10 --- Construction of Full Length Mas cDNA into pBluescript® II SK Vector --- p.38 / Chapter 2.2.11 --- Southern Blot Analysis / Chapter 2.2.11.1 --- Preparation of DIG-labeled Mas Probe --- p.38 / Chapter 2.2.11.2 --- Enzyme Restriction of Genomic DNA --- p.39 / Chapter 2.2.11.3 --- Transferring DNA to Nylon Membrane --- p.40 / Chapter 2.2.11.4 --- Prehybridization and Hybridization --- p.40 / Chapter 2.2.11.5 --- Post-hybridization Washes and Blocking --- p.41 / Chapter 2.2.11.6 --- Detection --- p.41 / Chapter 2.2.12 --- DNA Sequencing / Chapter 2.2.12.1 --- Manual Sequencing --- p.42 / Chapter 2.2.12.2 --- Autosequencing --- p.43 / Chapter 2.2.12.3 --- Sequencing Primers --- p.44 / Chapter 2.2.13 --- Cell Culture --- p.45 / Chapter 2.2.14 --- Protein Assay by Modified Lowery --- p.46 / Chapter 2.2.15 --- SDS-PAGE and Western Blot Analysis --- p.47 / Chapter 2.3 --- Results --- p.49 / Chapter 2.4 --- Discussion --- p.60 / Chapter Chapter 3: --- Analysis of Transfected Mas Cell Lines / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Materials --- p.62 / Chapter 3.2.2 --- Cell Culture and Transfection / Chapter 3.2.2.1 --- Cell Culture --- p.62 / Chapter 3.2.2.2 --- Transfection Optimization --- p.62 / Chapter 3.2.2.3 --- Fluorescent SEAP Assay --- p.63 / Chapter 3.2.2.4 --- Transient Transfection --- p.64 / Chapter 3.2.2.5 --- Stable Cell Line Construction --- p.64 / Chapter 3.2.3 --- Protein Assay ESL --- p.65 / Chapter 3.2.4 --- SDS-PAGE and Western Blot Analysis --- p.65 / Chapter 3.2.5 --- Preparation of an AT1a Receptor Internal Standard for Quantitative RT-PCR Analysis / Chapter 3.2.5.1 --- Preparation of an AT1a Receptor cDNA by RT-PC --- p.66 / Chapter 3.2.5.2 --- Cloning of AT1A Receptor cDNA into pBluescript® II SK Vector --- p.67 / Chapter 3.2.5.3 --- Autosequence of pBluescript® II SK Vector/AT1AR --- p.68 / Chapter 3.2.5.4 --- Preparation of 100 bp Deleted AT1a Receptor cDNA by RT- PCR --- p.68 / Chapter 3.2.5.5 --- Cloning of Deleted AT1a R cDNA into pCAPs Vector --- p.71 / Chapter 3.2.6 --- Construction of Full Length Mas cDNA into pOPRSVI/MCS Operator Vector --- p.71 / Chapter 3.2.7 --- Preparation of an Mas Internal Standard for Quantitative RT-PCR Analysis / Chapter 3.2.7.1 --- Preparation of 100 bp Deleted Mas cDNA by RT- PCR --- p.72 / Chapter 3.2.7.2 --- Cloning of 100 bp Deleted Mas cDNA into pCAPs Vector (Mas/pCAPs) --- p.73 / Chapter 3.2.8 --- Quantitative RT-PCR Analysis of AT1A R Expression --- p.74 / Chapter 3.2.9 --- Quantitative RT-PCR Analysis for the Expression of Mas --- p.74 / Chapter 3.3 --- Results --- p.76 / Chapter 3.4 --- Discussions --- p.100 / Chapter Chapter 4: --- Cloning of AT1A Receptor Promoter / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Materials --- p.105 / Chapter 4.2.2 --- Genomic DNA Extraction From Rat Pancreas --- p.105 / Chapter 4.2.3 --- "Nest PCR Amplification of 3.2, 2.8 and 1.4kb AT1a Receptor Promoter" --- p.105 / Chapter 4.2.4 --- PCR Amplification of 2.2 kb Aproximal Portion of AT1a Receptor Promoter --- p.107 / Chapter 4.2.5 --- Construction of PCR Fragment of Angiotensin Receptor Promoter into Various Vector --- p.108 / Chapter 4.2.5.1 --- pSEAP2-Basic --- p.108 / Chapter 4.2.5.2 --- pBluescript® II SK Vector --- p.109 / Chapter 4.2.5.3 --- PCR Cloning Kit (pCAPs vector) --- p.109 / Chapter 4.2.5.4 --- PCR-TRAP Cloning System --- p.109 / Chapter 4.2.6 --- Direct PCR Analysis --- p.110 / Chapter 4.2.7 --- Autosequencing of PCR Fragment of AT1A Receptor Promoter --- p.111 / Chapter 4.3 --- Results --- p.114 / Chapter 4.4 --- Discussions --- p.130 / Chapter Chapter 5: --- General Discussion --- p.131 / Chapter Appendix 1 --- Composition of Solutions --- p.133 / Chapter Appendix 2 --- Published Abstract --- p.141 / References --- p.142

Activating mutations of the kras proto-oncogene are found in more than 90% of human pancreatic ductal adenocarcinoma (PDAC) and can result in increased activity of the NF-κB pathway, leading to constitutive production of proinflammatory cytokines such as TNF-α. Pancreatic cancer progression occurs through a series of pre-invasive lesions, pancreatic intraepithelial neoplasias (PanIN lesions), which progress into invasive carcinoma. The aim of this thesis is to understand the autocrine role of TNF-α produced by premalignant epithelial cells in pancreatic tumour progression. This cytokine has already been shown to be involved in the progression of cancer. The major hypothesis therefore tested was that TNF-α secreted by pre-malignant epithelial cells promotes the early stages of pancreatic carcinogenesis by sustaining an inflamed microenvironment. In the spontaneous kras+/LSL-G12D; pdx1-cre mouse model of pancreatic cancer, concomitant genetic deletion of the TNF-α/IKK2 pathway substantially delayed pancreatic cancer progression and resulted in downregulation of the classical Notch target genes hes1 and hey1. Cell lines from the different PanIN bearing mice were established and used to dissect the cooperation between TNF-α/IKK2 and Notch signalling during PanIN progression. Optimal expression of Notch target genes was induced upon TNF-α stimulation of the canonical NF-κB signalling pathway, in cooperation with basal Notch signals. Mechanistically, TNF-α stimulation resulted in phosphorylation of histone H3 at the hes1 promoter and this signal was lost upon ikk2 genetic deletion. HES1 suppressed the expression of pparg, which encodes for the anti-inflammatory nuclear receptor PPAR-γ. Thus, crosstalk between TNF-α/IKK2 and Notch sustained an intrinsic inflammatory profile of the transformed cells. The treatment of PanIN bearing mice with rosiglitazone, a PPAR-γ agonist, also delayed PanIN progression. A malignant cell-autonomous, low-grade inflammatory process was shown to operate from the very early stages of kras-driven pancreatic carcinogenesis, which may cooperate with the Notch signalling pathway to promote pancreatic cancer progression.

Pancreatic ductal adenocarcinomas (PDACs) are highly aggressive malignancies, associated with poor clinical prognosis and limited therapeutic options. The KRAS oncogene is mutated in over 90% of PDACs and plays a pivotal role in tumor progression. Global gene expression profiling of PDAC reveals 3-4 major molecular subtypes with distinct phenotypic traits and pharmacological vulnerabilities, including variations in oncogenic KRAS pathway dependencies. PDAC cell lines of the aberrantly differentiated endocrine exocrine (ADEX) subtype are robustly KRAS-dependent for survival. The KRAS gene is located on chromosome 12p11-12p12, a region amplified in 5-10% of primary PDACs. Within this amplicon, we identified co-amplification of KRAS with the STK38L gene in a subset of primary human PDACs and PDAC cell lines. This provided rationale to determine whether PDAC cell lines are dependent on STK38L expression for proliferation and viability. STK38L (also known as NDR2) encodes a nuclear Dbf2-related (NDR) serine/threonine kinase, which shares homology with Hippo pathway LATS1/2 kinases. We show that STK38L expression levels are elevated in a subset of primary PDACs and PDAC cell lines that display ADEX subtype characteristics, including overexpression of mutant KRAS. RNAi-mediated depletion of STK38L in a subset of ADEX subtype cell lines results in decreased cellular proliferation and increased apoptotic cell death. Concomitant with cytostatic and cytotoxic effects, STK38L depletion causes increased expression of the LATS2 kinase and the cell cycle regulator p21. LATS2 depletion partially rescues the cell proliferation and viability effects of STK38L depletion. Lastly, high STK38L mRNA expression is associated with worse patient prognosis compared to low STK38L expression in PDACs. Taken together, our study uncovers STK38L as a candidate, targetable vulnerability in a subset of molecularly defined PDACs. / 2019-11-01T00:00:00Z

The Wnt signaling pathway is highly conserved and participates in many important cellular functions including differentiation, embryonic development and tissue generations. £]-catenin, the central mediator of the Wnt signaling, interacts with the TCF/LEF family of transcription factors in the nucleus and initiates downstream gene transcription. In addition, £]-catenin is known as a proto-oncogene implicated in numerous cancers including colorectal, cervical, endometrial and skin cancer. Chibby (Cby) is evolutionarily conserved in many species and acts as a repressor of Wnt/£]-catenin signaling. In our previous study, we have established that Cby over-expression attenuated £]-catenin translocation to nucleus and its transcriptional activity. Thus, it was hypothesized that Cby may possess potential tumor suppressing capabilities. In the present study, we first explored endogenous Cby expression status in human cervical cancer cells: HeLa and SiHa cell lines. It was observed that Cby mRNA and protein levels were significantly down-regulated in both cancer lines compared with primary cervical cells. We then conducted functional assays of tumorigenicity on both cells using adenoviral-encoded Cby and its NLS (nuclear localization signaling) deleted variant (Cby∆NLS). It was found that gene delivery of Cby or Cby∆NLS inhibited the proliferation, invasiveness, and colony forming in HeLa and SiHa cells. Immunofluorescent analysis revealed that Cby or Cby∆NLS gene transfer reduced the expression of Ki-67, a cell proliferative marker. Furthermore, Cby or Cby∆NLS restoration induced apoptosis and perturbed cell cycle progression in both cervical cancer cells. Finally, Cby over-expression decreases the expression of £]-catenin/TCF4 regulated genes such as c-myc and PCNA, which might contributed to the anti-neoplastic mechanism for Cby in cervical cancer cell lines. Our results strongly suggest that Cby may serve as a tumor suppressor gene during cervical carcinogenesis, and may facilitate in creation of new therapeutic methods.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 77-99).

Deciphering genetic determinants of tumorigenesis is the greatest challenge and promise of the present-day era of biomedical research. As extensive tumor genome characterization efforts of the past decade had revealed, tumor genomes harbor multiple point mutations and gene copy number alterations. This exquisite complexity brings forth the challenge of distinguishing numerous incidental alterations from those that are functionally relevant to tumorigenesis. During the past decade, functional genetic screens have shown their utility in identifying genetic changes that functionally contribute to tumor-specific hallmarks and thus hold a great potential for identifying promising new targets for the rational design of successful anticancer therapies. A key hallmark of cancer cells is their ability to escape signals that govern homeostasis of normal tissue. In normal epithelia, growth and survival of cells is dictated by their physical anchorage to the extracellular matrix, and disruption of proper cell-matrix anchorage triggers cell death. Tumors of epithelial origin develop ways to subvert anoikis signals, which enables both their uncontrollable expansion at the primary site as well as metastatic colonization of distant organs. Understanding the genetic determinants of matrix-independent growth of cancer cells is a promising approach to identify potent and selective anticancer targets. In the work presented in this dissertation, we use an unbiased functional genetic screening approach to test a large set of eight thousand human genes to identify those that are involved in inducing and maintaining resistance of mammary epithelial cells to matrix detachment-induced cell death. We show that a cell adhesion molecule PVRL4 promotes cell survival in the absence of matrix anchorage in normal epithelial cells and in cancer cells. Our work reveals that PVRL4 promotes anchorage-independent growth by promoting cell-to-cell attachment and matrix-independent c-Src activation. PVRL4 is focally and frequently amplified in several types of solid tumors. Growth of orthotopically implanted tumors in vivo is inhibited by blocking PVRL4-driven cell-to-cell attachment with monoclonal antibodies, demonstrating a novel strategy for targeted therapy of cancer.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate the expression of mRNA targets, and are aberrantly expressed in several cancers, including breast cancer. Using a transgenic mouse model of mammary tumorigenesis (MTB-IGFIR), a miRNA array was previously performed in our lab to study the expression level of various miRNAs in mammary tumours compared to wild-type mammary tissue. Next, the expression of a number of the differentially expressed miRNAs was confirmed and manipulated in a tumour cell line (RM11A) generated from a MTB-IGFIR mammary tumour. Synthetic miRNA precursors and inhibitors were then used to overexpress and knockdown, respectively, the levels of five miRNAs: miR-31, miR-183, miR-200c, miR-210, and miR-378. Upon optimization of miRNA overexpression and downregulation in RM11A cells, this study tested the effects of these miRNAs on cellular growth, survival, or invasiveness in vitro. Compared to negative controls, overexpression of all five miRNAs was associated with a significant decrease in cellular invasion, while only the overexpression of miR-31 had a significant effect on proliferation. No significant effects were found on cell survival. Our results implicate these five miRNAs in different aspects of mammary tumorigenesis, as well as having a tissue specific role in RM11A cells.