Dissertations / Theses on the topic '111201 Cancer Cell Biology'
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1
Bair, Elisabeth Laurine. "Cell-cell and cell-matrix interactions involved in cancer invasion." Diss., The University of Arizona, 2004. http://hdl.handle.net/10150/280673.
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In order for a cancer to metastasize, it must first invade through the basement membrane that surrounds it, invade blood vessels and travel through the bloodstream to a new location where it extravasates the vessel and begins growing at the new site. The mechanisms by which a cancer becomes able to invade and metastasize are currently under intense study. Interactions of the cell with its environment via cell-cell contacts, extracellular matrix (ECM) interactions, and circulating proteins are thought to play a major role in signaling for these invasive processes to occur. Upregulation of proteolytic enzymes, such as the matrix metalloproteases, is suspected of being involved in the metastatic process. Cell-cell and cell-matrix contacts via integrins and cadherins are necessary for upregulation of the matrix metalloprotease matrilysin in oral squamous cell carcinoma. In an effort to identify the factors involved in upregulation of matrilysin expression detected in a co-culture of oral squamous cell carcinoma (SCC) cells and fibroblast cells, a coculture model designed to represent the actual tumor environment, we show that inhibition of beta1 integrin, E-cadherin, and N-cadherin with blocking antibodies thoroughly decreases the induction of matrilysin in the co-culture model. This demonstrates that interactions between cancer cells and normal cells surrounding them may allow for invasion and metastasis. The protein 90K may also play a role in the invasive process of prostate cancer. It functions as an immune modulator upregulating cytokines that induce MMPs and we show that it can induce matrilysin expression in prostate cancer cells. It also functions in cell aggregation, which can help cells survive during metastasis. For this reason, expression of 90K in prostate cancer, which we examined, may be indicative of aggressive disease, making 90K a potentially useful tumor marker. Cell-matrix contacts are also important for the transmembrane matrix metalloprotease MT1-MMP cleavage of laminin-10. We demonstrate that recombinant MT1-MMP is able to cleave human laminin-10 into four distinct products. This allows for prostate cancer cell migration on laminin-10 coated substrates, which can be inhibited with the addition of MT1-MMP antisense oligonucleotides. Ln-10 cleavage also occurs in vivo in human prostate tissue, indicating that this cell-matrix interaction has in vivo relevance in human prostate cancer.
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Krubasik, Davia Regina Editla. "The role of Metalloproteinases in cancer cell biology." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612722.
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Ng, Sheng Rong. "CRISPR-mediated interrogation of small cell lung cancer." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/117782.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged student-submitted from PDF version of thesis. Vita.
Includes bibliographical references.
Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine lung carcinoma that remains among the most lethal of solid tumor malignancies. Despite decades of research, treatment outcomes for SCLC remain very poor, highlighting the need for novel approaches to target the disease. Recent genomic sequencing studies have identified multiple recurrently altered genes in human SCLC tumors, many of which remain to be functionally validated. Genetically engineered mouse models (GEMMs) of SCLC have been developed that recapitulate many key features of human SCLC. These models have been used extensively to investigate various aspects of SCLC biology, including tumor initiation, progression and metastasis. The development of the CRISPR-Cas9 system has greatly facilitated genome editing in mammalian cells, leading to its widespread adoption for various applications in cancer biology. We have utilized this system in two complementary ways to investigate the molecular mechanisms involved in SCLC initiation, progression and maintenance. Firstly, we have adapted the CRISPR-Cas9 system for use in GEMMs of SCLC, to enable rapid modeling and functional validation of candidate tumor suppressor genes in vivo. Using this system, we have demonstrated that p107, a member of the retinoblastoma family that is mutated in a significant fraction of human SCLC tumors, is a functional tumor suppressor in SCLC. Notably, loss of p107 in SCLC tumors resulted in significant phenotypic differences compared with loss of its close relative, p130. We also demonstrated that CRISPR-induced mutations can be used to infer lineage relationships between primary and metastatic tumors in the same animal. Secondly, we have performed a CRISPR-based genetic screen, utilizing a custom sgRNA library targeting the druggable genome, to identify novel SCLC-specific genetic vulnerabilities. We found that SCLC cells displayed enhanced sensitivity towards disruption of several key metabolic pathways, including the de novo pyrimidine biosynthesis pathway. Pharmacological inhibition of Dhodh, a key enzyme in this pathway, reduced the viability of SCLC cells in vitro and strongly suppressed SCLC tumor growth in vivo, validating this pathway as a promising therapeutic target in SCLC. Taken together, the work presented here demonstrates the utility of the CRISPR-Cas9 system for performing functional interrogation of SCLC.
by Sheng Rong Ng.
Ph. D.
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Kay, Sophie Kate. "Cell fate mechanisms in colorectal cancer." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:f19bf73d-0c0e-4fff-9589-bf43f9ff12f0.
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Colorectal cancer (CRC) arises in part from the dysregulation of cellular proliferation, associated with the canonical Wnt pathway, and differentiation, effected by the Notch signalling network. In this thesis, we develop a mathematical model of ordinary differential equations (ODEs) for the coupled interaction of the Notch and Wnt pathways in cells of the human intestinal epithelium. Our central aim is to understand the role of such crosstalk in the genesis and treatment of CRC. An embedding of this model in cells of a simulated colonic tissue enables computational exploration of the cell fate response to spatially inhomogeneous growth cues in the healthy intestinal epithelium. We also examine an alternative, rule-based model from the literature, which employs a simple binary approach to pathway activity, in which the Notch and Wnt pathways are constitutively on or off. Comparison of the two models demonstrates the substantial advantages of the equation-based paradigm, through its delivery of stable and robust cell fate patterning, and its versatility for exploring the multiscale consequences of a variety of subcellular phenomena. Extension of the ODE-based model to include mutant cells facilitates the study of Notch-mediated therapeutic approaches to CRC. We find a marked synergy between the application of γ-secretase inhibitors and Hath1 stabilisers in the treatment of early-stage intestinal polyps. This combined treatment is an efficient means of inducing mitotic arrest in the cell population of the intestinal epithelium through enforced conversion to a secretory phenotype and is highlighted as a viable route for further theoretical, experimental and clinical study.
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Neal, Corey Lekeil. "Snail mediates epithelial mesenchymal transition and cell adhesion in human prostate cancer cell lines." DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 2011. http://digitalcommons.auctr.edu/dissertations/233.
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Snaill (Snail) transcription factor induces Epithelial Mesenchymal Transition (EMT), in which epithelial cells down-regulate cell adhesion genes such as E-cadherin and up-regulate mesenchymal genes such as vimentin, leading to increased invasion and migration. Maspin is a putative tumor suppressor that is down-regulated in breast and prostate cancer and has been associated with decreased cell motility, while Snail is increased in breast cancer and associated with increased tumor motility and invasion. Very little is known about the role of Snail in cellular adhesion to the extracellular matrix (ECM) and its role in regulation of maspin expression has not been explored. We hypothesized that Snail will lead to decreased cellular adhesion to the extracellular matrix through integrin regulation, concomitant with increased cell migration. Our studies showed that Snail decreases cell adhesion to fibronectin (FN) and collagen I (CGN) matrix through inhibition of cL5 (fibronectin receptor), c~2 (collagen receptor), ~3 1 integrins, while migration to FN and CON was increased. We have also identified an inverse relationship between Snail and rnaspin in normal prostate epithelial cells and prostate cancer cells and shown for the first time that Snail can inhibit maspin expression.
This work utilized normal prostate epithelial cells (PrEC), androgen-dependent LNCaP cells, androgen-independent C4-2, DU145, 22Rvl, ARCaP and PC3 prostate cancer cell lines. Cells with either the endogenous, overexpression or knockdown of the Snail transcription factor were utilized to observe the role of Snail in cell adhesion and migration and to establish its molecular mechanism(s) of action. We have provided direct evidence that the Snail transcription factor negatively impacts prostate cancer cell adhesion and migration to fibronectin and collagen matrices. This activity was regulated through integrins and the mitogen-activated protein kinase (MAPK) signaling pathway. Additionally, we have shown that Snail negatively regulates maspin expression by inhibiting activity at the maspin promoter.
Collectively, these studies define a new role for Snail in cell adhesion to the ECM. Therefore, targeting of Snail may be useful to re-induce expression of maspin putative tumor suppressor, increase cell adhesion to ECM, decrease cell migration and prevent prostate cancer tumor progression and metastasis.
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McNae, Fiona. "The cell biology of non-genotoxic hepatocarcinogens." Thesis, University of Surrey, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260350.
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Weitzel, Douglas H. "Modulation of cell cycle checkpoints by anti-cancer agents /." The Ohio State University, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=osu148820531851081.
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Horlock, Nigel. "The cell biology of basal cell carcinoma : relationship to histology and clinical outcome." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391606.
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Poluri, Raghavendra Tejo Karthik. "Using bioinformatic analyses to understand prostate cancer cell biology." Master's thesis, Université Laval, 2020. http://hdl.handle.net/20.500.11794/66803.
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Le cancer de la prostate (CaP) affecte 1 homme sur 7 au cours de sa vie. C’est le cancer numéro un diagnostiqué chez l'homme. Il s'agit du quatrième cancer le plus fréquent au Canada. Le CaP est une maladie hormonodépendante diagnostiquée chez l'homme. Les androgènes jouent un rôle vital dans la progression de la maladie. La première ligne de traitement, suivant une intervention chirurgicale ou un traitement de radiothérapie, est la thérapie de déprivation aux androgènes. Malgré une réponse initiale positive à l'inhibition des androgènes, la progression de la maladie vers un cancer de la prostate résistant à la castration (CRPC) est presque inévitable. Aux différentes étapes du CaP, le récepteur des androgènes joue un rôle majeur. Ainsi, cette thèse décrit les méthodes développées et utilisées pour mieux comprendre la biologie du CaP et le rôle joué par les androgènes dans cette maladie. Le travail démontré dans cette thèse se compose principalement d'analyses bioinformatiques effectuées sur des ensembles de données accessibles au public et d'un « pipeline » construit pour analyser des données RNA-Seq. Un pipeline RNA-Seq a été développé pour comprendre l'impact des androgènes et des gènes régulés lors du traitement aux androgènes dans les modèles de cellules de CaP. Ce pipeline bioinformatique se compose de divers outils qui ont été décrits ci-dessous dans le chapitre 1. L'objectif principal de ce projet était de développer un pipeline pour analyser les données RNA-Seq qui aide à comprendre et à définir les voies et les gènes métaboliques qui sont régulés par les androgènes, et qui jouent un rôle important dans la progression du CaP. Le flux de travail expérimental consistait en deux lignées cellulaires positives aux récepteurs aux androgènes LNCaP et LAPC4. Toutes les données utilisées dans ce projet ont été rendues publiques pour que la communauté de recherche puisse effectuer diverses autres études et analyses comparatives pour comprendre les fonctions des androgènes dans un sens beaucoup plus profond afin de développer de nouvelles thérapies pour traiter le CaP. Dans un autre projet décrit au chapitre 2, des analyses bioinformatiques ont été réalisées sur des données accessibles au public pour comprendre la fréquence de la perte et de l'altération génomique du gène PTEN localisé à 10q23. Ces analyses ont mis en évidence la fréquence d'altération génomique de PTEN qui est beaucoup plus élevée dans le CRPC que dans le CaP localisé. Ces analyses ont également aidé à identifier d'autres gènes altérés dans le CaP. Ces gènes n’ont pas été beaucoup étudiés dans la littérature, mais il semble que certains d’entre eux possèdent des caractéristiques de suppresseurs de tumeurs. Ces résultats pourraient être un bon début pour des analyses plus approfondies concernant la perte de gènes.La compréhension des fonctions de AR et de la suppression de PTEN aidera à développer de nouvelles stratégies et approches pour diagnostiquer et traiter le CaP. L'intégration des analyses bioinformatiques à la recherche clinique ouvre une nouvelle perspective dans le domaine de la recherche du CaP.Prostate Cancer (PCa) affects 1 in 7 men in their lifetime and is the number one diagnosed cancer in men. It is the 4th most common cancer in Canada. PCa is a hormone-dependent disease diagnosed in men. Androgens play a vital role in the disease progression. The standard of care to treat PCa, following surgery or radiation therapy, is the androgen deprivation therapy (ADT). In spite of initial positive response to androgen inhibition, the progression of the disease to castration-resistant prostate cancer (CRPC) is almost inevitable. Across the various stages of PCa, the androgen receptor (AR) plays a major role. This thesis portrays the methods developed and used to understand PCa biology. The work demonstrated in this thesis majorly consists of bioinformatic analyses performed on publicly available data sets and a pipeline built to analyse RNA-Seq data. An RNA-Seq pipeline has been developed to understand the impact of androgens and the genes regulated upon androgen treatment in PCa cell models. This bioinformatic pipeline consists of various tools which have been described below in chapter 1. The major goal of this project was to develop a pipeline to analyse the RNA-Seq data which helps to understand and define the metabolic pathways and genes regulated by androgens which play an important role in PCa disease progression. The experimental workflow consisted of two androgen receptor positive cell lines LNCaP and LAPC4. All the data used in this project has been made publicly available for the research community to perform various other comparative studies and analyses to understand the functions of androgens in a much deeper sense to develop novel therapies to treat PCa. In another project described in chapter 2, bioinformatic analyses have been performed on publicly available data to understand the loss and genomic alteration frequency of the gene PTEN occurring at 10q23. These analyses highlighted that the genomic alteration frequency of PTEN is much higher in CRPC than in localised PCa, and also helped in identifying other genes which are lost along with PTEN. The lost genes have not been studied much in literature, but few studies demonstrated that they might possess tumor suppressor characteristics. These results might be a good start for further deeper analyses regarding the lost of genes. Understanding the functions of AR and the deletion of PTEN will help for the development of novel strategies and approaches to diagnose and treat PCa. Integration of bioinformatic analyses with clinical research open up a new perspective in the PCa research domain.
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Popovic, Predrag. "Cisplatin resistance in nonsmall cell lung cancer: Role of platinum accumulation and cell membranes." Thesis, University of Ottawa (Canada), 1994. http://hdl.handle.net/10393/9662.
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A cisplatin resistant cell line named E-8/0.7 was derived from the nonsmall cell lung cancer HTB 56 cell line. According to IC$\sb{50}$ estimations, E-8/0.7 cells were approximately 7.7 times more resistant to cisplatin than were HTB 56 cells. Uptake of cisplatin 100 $\mu$M over 1 hour was measured by atomic absorption spectrophotometry, and showed around a 30% lower uptake in E-8/0.7 cells compared to HTB 56 cells. Barotropic behaviour was analyzed by pressure tuning infrared spectroscopy. In the CH symmetric stretching region, we found a lower break point (and consequently lower membrane fluidity) in E-8/0.7 cells compared to the parent cell line. Lipid analyses of the two cell lines showed a higher cholesterol to phospholipid molar ratio in E-8/0.7 than in HTB 56 cells. Moreover, concentration of sphingomyelin was higher in E-8/0.7 cells than in HTB 56 cells.
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Dassie, Justin Patrick. "Selective targeting of cancer cells with RNA aptamers." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/1310.
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Two of the most commonly diagnosed malignancies in men and women are cancers of the prostate and breast, respectively. Though many advances have been made in reducing the overall morbidity and mortality associated with these diseases, the high number of deaths that still occur emphasizes the need for safer and more effective therapeutic options. To this end, our lab was the first to describe the use of RNA aptamers to specifically deliver cytotoxic siRNAs to PSMA positive prostate cancer cells. This reagent, termed an aptamer-siRNA chimera, was shown to be an effective targeted cancer therapeutic upon intratumoral injection in a pre-clinical, xenograft, mouse model of prostate cancer. However, further work was needed to realize the full clinical potential of RNA aptamer-siRNA chimeras as a targeted therapeutic modality.
The thesis laid out herein, describes work performed to optimize aptamer-siRNA technology in order to enable clinical translation and to increase the scope of this technology (i.e. increase the cancer types for which this technology can be used). We describe several improvements to our first generation PSMA aptamer-siRNA chimera which, include: decreasing the overall nucleotide content to aid in chemical synthesis, altering the siRNA structure to improve RNAi processing and addition of a 20kDa PEG moiety to increase pharmacokinetics/pharmacodynamics. All of these modifications lead to a more effective reagent at lower doses. Importantly, we demonstrate that our optimized reagent is now effective upon systemic administration in an in vivo mouse model of prostate cancer. In addition, we have also identified new aptamers to the receptor tyrosine kinase (RTK) EphA2. Given the broad expression of this RTK on various cancers, this work seeks to extend the scope of targeted aptamer therapeutics beyond that of prostate cancer. Finally, we demonstrate a novel aptamer selection methodology termed cell-internalization SELEX. This approach allowed us to select for aptamers that specifically targeted and internalize into HER2 expressing cells. This allowed us to readily translate all identified aptamers into aptamer-siRNA chimeras. We show that all chimeras tested were able to sensitize HER2+ breast cancer cells to low- dose cisplatin treatment.
Taken together, the work described in this thesis significantly advances the field of targeted cancer therapeutics. Importantly, by demonstrating cancer cell-specific delivery of siRNA, our technology overcomes one of the most significant hurdles to the therapeutic use of siRNAs, delivery.
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凌明達 and Ming-tat Patrick Ling. "A study of molecular and cell biology of prostate tumorigenesis in cell culture." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31223102.
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Dremann, David Michael. "Pluronic Activity in Hyperthermia-induced Cancer Cell Death." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1247425426.
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Brauneis, Alison Dooley. "Investigating the initiation and progression of small cell lung cancer." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/63063.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.Cataloged from PDF version of thesis.
Includes bibliographical references.
Small cell lung cancer (SCLC) comprises 18% of all lung cancer cases and is an aggressive disease with a five-year survival rate of less than 5%, mainly due to the advanced nature of the disease at the time of diagnosis. Despite the need to better understand this disease, the genetic lesions that contribute to SCLC remain poorly characterized. To investigate the genetic aberrations that occur in SCLC, we analyzed the copy number alterations in tumors and metastases arising in a mouse model of SCLC (mSCLC), driven by conditional inactivation alleles of two tumor suppressor genes, Trp53 and Rbl. We identified frequent, high-level amplification of a novel, protooncogenic transcription factor Nuclear Factor I/B (Nfib in mouse, NFIB in human), which frequently occurred coincident with amplification of a previously characterized oncogene, L-myc (Mycl). Functional studies revealed cooperation between Nfib and Lmyc in cellular transformation. Comparative genomics identified NFIB amplifications in human SCLC and uncovered a role for NFIB in regulating cell viability and proliferation. We also examined the effect of lung injury on SCLC initiation and progression utilizing naphthalene, a chemical that ablates the cells that line the bronchioles of the lung. The pulmonary neuroendocrine cells, the putative cell of origin of SCLC, are refractory to naphthalene-mediated injury. We demonstrated that naphthalene-mediated injury prior to tumor initiation in the mouse model of SCLC induced more advanced mSCLC lesions and decreased tumor latency. Throughout the course of this thesis work, we successfully utilized a mouse model of SCLC to interrogate the initiation of SCLC as well as to define the genetic alterations that occur in SCLC tumors and metastases. This work has led to the identification of candidate genes that promote tumor progression and to a better understanding of the process of tumor initiation. We anticipate that these findings will not only enhance our understanding of SCLC, but may lead to the development of therapeutics used to treat this aggressive disease.
by Alison L. Dooley.
Ph.D.
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Valenciaga, Anisley. "Cell cycle regulators and transcriptional targeting in Medullary Thyroid Cancer." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523891651533415.
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Liu, Yunpeng Ph D. Massachusetts Institute of Technology. "Integrative multi-omics dissection of cancer cell states and susceptibility." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/130818.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February, 2021Cataloged from the official PDF of thesis. "February 2021."
Includes bibliographical references (pages 217-239).
Cancer cells are characterized by a broad spectrum of unique genetic, epigenetic and transcriptional states, which are often concomitant with high degrees of plasticity in cell identity. These cell states and the fluidity therein are a major source of resistance to both chemotherapy and targeted therapy. Combinatorial efforts in experimental assays and computational modeling are pivotal for understanding the origins of cancer cell plasticity and exposing cell state-specific vulnerabilities. In this thesis, I will first present my studies on two clinically challenging types of hematopoietic malignancies and discuss key genes that sustain cell identity and survival programs revealed through multi-omics approaches.
In the first study, a combination expression, chromatin binding and chromatin accessibility analyses revealed the plant homeodomain finger-like family protein PHF6's novel functions as a lineage identity regulator in a mouse model of BCR-ABL-driven B cell acute lymphoblastic leukemia. In the second case, single cell transcriptomic profiling, computational inference of cell cycle trajectories and unbiased functional genomics jointly identified RAD51B as a uniquely essential gene in near-haploid leukemia. Finally, to systematically model heterogeneous cell states and generate readily testable predictions of susceptibilities in cancer, I proposed a novel computational pipeline that integrates multiple data types to construct a quantitative model of transcription regulation, which can in turn be used to infer changes in gene expression in response to transcription factor perturbation.
The pipeline then uses these gene expression responses to perturbations to estimate changes in protein activity and finds a combination of protein activity score changes that best predicts changes cell fitness. Applying the pipeline to glioblastoma multiforme - a cancer type that lacks effective targeted therapy, I prioritized a small set of genes including MYBL2 as subtype-specific candidate targets. My thesis work demonstrates the power of integrative, multi-omics approaches for effective discovery of susceptibilities in cancer and highlights an emerging paradigm for understanding the information flow in the cellular circuitry.
by Yunpeng Liu.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
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Zeid, Rhamy. "Characterization and Disruption of Cis Regulatory Elements in Cancer." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493536.
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Enhancers are cis regulatory elements that play key roles in the control of cell-type specific gene expression programs. In cancer, enhancer deregulation plays a key role in maintaining gene regulatory programs that underlie an oncogenic state. This dissertation focuses on understanding and modulating aberrant enhancer activity to identify potential vulnerabilities in human cancers. These studies were empowered by evolving technologies in genome-wide measurements of enhancer factors, computational approaches, and chemical and genetic tools to disrupt enhancer function.
In high-risk pediatric neuroblastoma, the transcription factor MYCN is frequently amplified and treatment options for these patients are largely ineffective thus establishing the need for improved therapeutic options. To identify previously unrecognized dependencies in neuroblastoma, we generated genome-wide maps of the active enhancer gene regulatory landscape leading to the identification of ID1 as an uncharacterized dependency in neuroblastoma. These results outline a strategy to identify alternative therapeutic avenues based on a holistic understanding of aberrant enhancer activity.
While MYCN amplification is the defining feature of high-risk neuroblastoma, a detailed mechanistic understanding of oncogenic transcriptional rewiring has been stalled by a lack of genome-wide binding data.
Here we present the dynamic and temporally resolved landscape of genome-wide MYCN occupancy in neuroblastoma. We find that deregulated MYCN binding at enhancers (termed enhancer invasion) is critical to maintaining the oncogenic station and identify the lineage specific transcription factor TWIST1 as a key collaborator and synthetic lethality of oncogenic MYCN. These data suggest that MYCN enhancer invasion shapes transcriptional amplification in neuroblastoma to promote tumorigenesis.
The development of small molecule inhibitors of the bromodomain and extra-terminal (BET) family of proteins provides a pharmacological strategy to inhibit enhancer activity. The efficacy of BET inhibition in several cancers has prompted efforts to predict and understand mechanisms of resistance to BET inhibition. Here, we use a newly developed class of small molecules to pharmacologically induce targeted degradation of the BET family. In triple negative breast cancer, we demonstrate that targeted BET family degradation effectively overcomes BET inhibitor resistance. These studies suggest BET degradation as a strategy to overcome BET inhibitor resistance and further disrupt and dissect enhancer activity in cancer.Medical Sciences
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Wright, Muelas Marina. "A systems biology approach to cancer metabolism." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/a-systems-biology-approach-to-cancer-metabolism(27286c8a-0281-4256-b749-2ec9bd36370f).html.
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Cancer cells have been known for some time to have very different metabolismas compared to that of normal non proliferating cells. As metabolism is involvedin almost every aspect of cell function, there has been a recent resurgence ofinterest in inhibiting cancer metabolism as a therapeutic strategy. Inhibitors thatspecifically target altered metabolic components in cancer cells are being developedas antiproliferative agents. However, many such inhibitors have not progressedinto the clinic due to limited efficacy either in vitro or in vivo. In this study weexplore the hypothesis that this is often due to the robustness of the metabolicnetwork and the differences between individual cancer cell lines in their metaboliccharacteristics. We take a systems biology approach. We investigate the cellular bioenergetic profiles of a panel of five non-small celllung cancer cell lines before and after treatment with a novel inhibitor of theglutaminase-1 (GLS1) enzyme. Additionally, we explore the effects of this inhibitoron intracellular metabolism of these cell lines as well as on the uptake and secretionof glucose, lactate and amino acids. To be able to do the latter robustly, wehad to modify the experimental assay considerably from procedures that seemto be standard in the literature; using these earlier procedures the metabolicenvironment of the cells was highly variable, leading to misleading results onthe metabolic effects of the inhibitor. We reduced cell density, altered mediumvolume and changed the time-window of the assay. This led to the cells growingexponentially, appearing indifferent to the few remaining changes. In this newassay, the metabolic effects of the glutaminase inhibitor became robust. One of the most significant results of this study is the metabolic heterogeneitydisplayed across the cell line panel under basal conditions. Differences in themetabolic functioning of the cell lines were observed in terms of both theirbioenergetic and metabolic profile. The amount of respiration attributed tooxidative phosphorylation differed between cell lines and respiratory capacity wasattenuated in most cells. However, the rate of glycolysis was similar betweencell lines in this assay. These results suggest that the Warburg effect arisesthrough a greater diversity of mechanisms than traditionally assumed, involvingvarious combinations of changes in the expression of glycolytic and mitochondrialmetabolic enzymes. The effects of GLS1 inhibition on cellular bioenergetics and metabolism alsodiffered between cell lines, even between resistant cell lines, indicating that theremay also be a diversity of resistance mechanisms. The metabolomic response ofcell lines to treatment suggests potential resistance mechanisms through metabolicadaptation or through the prior differences in the metabolic function of resistantcell lines. Part of the metabolome response to GLS1 inhibition was quite specificfor sensitive cells, with high concentrations of IMP as the strongest marker. Our results suggest that the metabolome is a significant player in what determinesthe response of cells to metabolic inhibitors, that its responses differ between cancercells, that responses are not beyond systems understanding, and that thereforethe metabolome should be taken into account in the design of and therapy withanti-cancer drugs.
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Thor, Straten Per. "T-cell response against human malignant melanoma." Thesis, Open University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264405.
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Dowling, Ryan. "The role of mTOR signalling in translational control and cancer." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66869.
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Abstract The serine/threonine kinase mTOR plays a critical role in the regulation of cellular proliferation, growth, differentiation, autophagy, and cytoskeletal organization. Two mTOR containing complexes exist in mammalian cells: mTORC1 and mTORC2. mTORC1 mediates its effects through the control of mRNA translation initiation via phosphorylation of its two major downstream targets: the 4E-BPs and S6Ks. In order to study the individual roles of S6Ks and 4E-BPs downstream of mTORC1, we utilized new active-site mTOR inhibitors and cells deficient for either 4E-BPs or S6Ks. Surprisingly, we found that 4E-BPs and S6Ks play distinct roles in mediating the activity of mTORC1, with the 4E-BPs mediating cell proliferation and the S6Ks regulating cell growth. The effects of active-site mTOR inhibitors on cell proliferation, cell cycle progression, and protein synthesis were highly attenuated in cells lacking expression of 4E-BPs (4E-BP DKO), while these processes were significantly inhibited in wild-type cells. However, the growth of 4E-BP DKO cells was equally sensitive to active site mTOR inhibitors, as compared to their wild-type counterparts. In contrast, the growth of S6K1/2 null cells was refractory to the effects of active site mTOR inhibitors, whereas these cells were fully sensitive to the effects of the inhibitors on proliferation and cell cycle progression. These data support a model whereby the mTORC1 proliferative and growth signals diverge in mammalian cells to provide a system that allows for increased flexibility in the maintenance of full body homeostasis. mTOR is often over-activated in human cancers and as a result, has emerged as a target for anti-cancer therapies. The anti-diabetic drug metformin was recently identified as a potential anti-cancer agent. To elucidate its mechanism of action, we studied its effects on breast cancer cell proliferation, mTOR signalling and mRNA translation. In bRésumémTOR est une sérine/thréonine kinase qui joue un rôle primordial dans lecontrôle de plusieurs fonctions cellulaires, incluant la prolifération, la croissance, ladifférentiation, l'autophagie et la réorganisation du cytosquelette. Les cellulesmammifères possèdent deux complexes protéiques contenant mTOR, soit mTORC1 andmTORC2. mTORC1 exerce son action en contrôlant l'initiation de la traduction desARN messagers via la phosphorylation de ses principaux effecteurs : les 4E-BP et lesS6K. Dans le but de mieux comprendre les fonctions spécifiques des 4E-BP et des S6Ken aval de la signalisation cellulaire initiée par mTORC1, nous avons utilisé de nouveauxinhibiteurs spécifiques au site actif de mTOR, ainsi que des cellules n'exprimant pas les4E-BP ou les S6K. Nous avons découvert que les 4E-BP et les S6K jouent des rôlesdistinct en aval de mTORC1, les 4E-BP étant principalement impliquées dans laprolifération cellulaire, alors que les S6K jouent un rôle dans le contrôle de la croissancecellulaire. Les effets des inhibiteurs spécifiques au site actif de mTOR sur la proliférationcellulaire, la progression à travers le cycle cellulaire et la synthèse protéique sontfortement atténués dans les cellules nulles pour les 4E-BP, alors qu'ils sont inhibés demanière importante dans les cellules de type sauvage. Cependant, la croissance descellules 4E-BP nulles, en présence des inhibiteurs spécifiques au site actif de mTOR, estla même que celle observée pour les cellules de type sauvage. À l'opposé, la croissancedes cellules S6K nulles n'est pas affectée par ces inhibiteurs, tandis que les inhibiteursconservent leurs effets négatifs sur la prolifération et la progression au travers du cyclecellulaire dans ces mêmes cellules. Ces résultats sont en accord avec un modèle danslequel la signalisation en aval de mTORC1, pour promouvoir la prolifération et lacroissance cellulair
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Mireuta, Matei. "Aspects of insulin-like growth factor binding proteins in cancer." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114128.
Full textAbstract:
The insulin-like growth factor (IGF) system is composed of two ligands (IGF-1 and IGF-2), two receptors (IGF-1R and IGF-2R) and six binding proteins (IGFBP-1 to -6). IGFs act as endocrine, paracrine and autocrine growth factors and stimulate cell growth, proliferation and metabolism. There is extensive evidence, both from in vitro and in vivo models as well as population studies, that IGF physiology is relevant to neoplasia. IGF-1R is the physiologic receptor for both ligands and its activation elicits a plethora of changes at the cellular level, such as activation of PI3K/AKT/mTOR and Ras/Raf/MAP kinase pathways. Given its role in the maintenance and promotion of neoplasia, the IGF system represents a potential target in the context of cancer therapy.Classically, IGFBPs have been described as carrier proteins for IGFs in the blood and other fluids. They can regulate IGF bioavailability both positively through increases in ligand half-life as well as negatively through competition with the IGF-1R for ligand binding. In addition to their classical roles, there is evidence suggesting that IGFBPs can act independently of IGFs by poorly characterized mechanisms. Additionally, epidemiologic studies have correlated overexpression of certain IGFBPs, in particular IGFBP-2, with poor prognosis in various cancers.Although the role of IGFBPs has been extensively studied in the context of both normal and malignant growth, this thesis describes several new aspects of IGFBPs in neoplasia. In the second chapter, we study the effect of the PI3K/AKT/mTOR cascade on IGFBP-2 gene expression in a breast cancer cell line in vitro. We demonstrate that activation of this pathway essentially leads to an Sp1-dependent increase in IGFBP-2 gene transcription. We further show that Sp-1 is phosphorylated upon PI3K/AKT/mTOR pathway activation and accumulates in the nucleus. In the third chapter, we study the effects of 2-deoxyglucose (2-DG) on IGF-1:IGFBP-3 complex formation. A recent publication suggested that 2-DG unexpectedly disrupted IGF-1:IGFBP-3 binding leading to increases in IGF-1R and AKT signaling in various cell lines. We show by three different techniques that neither 2-DG nor glucose affect IGF-1:IGFBP-3 complex formation. We additionally show that the 2-DG effects observed are not consistent between cell lines and likely the result of changes in intracellular signaling. In the fourth chapter, we study the effects of a novel therapeutic antibody (BI836845) with high affinity for both IGF-1 and IGF-2. In mouse serum samples ex vivo, we show that the addition of BI836845 leads to a shift of IGF-1 from the IGFBPs to the antibody. In vivo, we demonstrate that BI836845 binds the vast majority of IGF-1. Finally, we demonstrate that BI836845 induces a decrease in IGFBP-3 and an increase in growth hormone levels in C57 BL/6 mice.L'ensemble du système de facteurs de croissance insulinomimétique (IGF) est composé de deux ligands (IGF-1 et IGF-2), de deux récepteurs (IGF- 1R et IGF-2R) et de six protéines de liaison (IGFBP-1 à 6). Les IGFs sont des hormones endocrines, paracrines et autocrines qui stimulent la croissance cellulaire, la prolifération et le métabolisme. Il existe un grand nombre d'études utilisant des approches épidémiologiques ou des modèles in vivo et in vitro qui démontrent l'importance des IGFs dans le contexte du cancer. Le IGF-1R est le récepteur physiologique des deux ligands et son activation mène à d'importants changements cellulaires tels que l'activation des voies de signalisation PI3K/AKT/mTOR et Ras/Raf/MAPK. Étant donné son rôle dans la promotion et dans la progression du cancer, le système des IGFs représente une cible potentielle pour le traitement du cancer. De façon classique, les protéines de liaison IGFBP ont été décrites comme de simples porteurs d'IGFs dans le sang et autres fluides. Les IGFBPs peuvent modifier la biodisponibilité des IGFs de façon positive en augmentant leur demi-vie ou de façon négative due à leur compétition avec le IGF-1R pour la liaison. En plus de leur rôle classique, il est de plus en plus évident que ces protéines peuvent agir de manière indépendante, mais les mécanismes impliqués restent flous. Également, il existe des études épidémiologiques qui ont corrélé la surexpression de IGFBPs, en particulier IGFBP-2, avec un pronostic défavorable dans plusieurs formes de cancer. Bien que le rôle des IGFBPs ait été largement étudié dans le contexte de la croissance normale et en néoplasie, la présente thèse révèle quelques nouveaux aspects de la physiologie des IGFBPs dans le contexte du cancer. En première partie, nous étudions l'effet de la voie de signalisation PI3K/AKT/mTOR sur l'expression du gène IGFBP-2 dans une lignée cellulaire de cancer du sein. Nous démontrons que l'activation de cette voie mène essentiellement à une augmentation de la transcription de ce gène de manière dépendante au facteur de transcription Sp-1. De plus, nous établissons que Sp-1 est phosphorylé par l'activation de la voie PI3K/AKT/mTOR et s'accumule dans le noyau. En deuxième partie, nous étudions les effets de la molécule 2-deoxyglucose (2-DG) sur la liaison entre IGF-1 et IGFBP-3. Un récent article avait suggéré un effet inhibitoire de cette molécule sur la formation de complexes IGF -1 :IGFBP-3. Nous démontrons par trois méthodes différentes que 2-DG ou la molécule apparentée glucose n'ont aucun effet sur la liaison entre IGF-1 et IGFBP-3. De plus, nous démontrons que les effets cellulaires de 2-DG sur l'activation de la voie PI3K/AKT/mTOR observées par les auteurs de l'article en question ne sont pas universels et sont probablement le résultat de signaux intracellulaires. Finalement, en dernière partie, nous étudions les effets d'un nouvel anticorps thérapeutique nommé BI836845 qui possède une grande affinité pour IGF-1 et IGF-2. Dans des échantillons de sérum de souris ex vivo, nous démontrons que l'ajout de BI836845 déplace IGF-1 des complexes naturels contenant les IGFBPs vers des complexes contenant l'anticorps. In vivo, nous démontrons que BI836845 lie la grande majorité d'IGF-1. Nous démontrons aussi que l'anticorps mène à une baisse de la concentration de IGFBP-3 et à une hausse de la concentration de l'hormone de croissance chez des souris C57 BL/6.
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Challa, Sridevi. "Mechanisms of IKBKE Activation in Cancer." Scholar Commons, 2017. http://scholarcommons.usf.edu/etd/6617.
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Cancer is the second leading cause of death in the USA and it is expected to surpass heart diseases making it important to understand the underlying mechanisms of cancer. The efforts to target single signaling molecule showed little success in increasing the patient survival and it can be due to increased compensation for cell survival by alternative pathway activations. Hence comprehensive understanding of the alternative signaling pathways may help us treat cancer better. Chronic inflammation is attributed to increased risk of cancer and emerging studies show the growing importance of both canonical and non-canonical IκB kinases such as IKKα, IKKβ, IKBKE and TBK1 in human cancer pathogenesis. Initially identified as activator of NFκB pathway, IKBKE was shown to play an important oncogenic role by regulating multiple pathways downstream. Although IKBKE is implicated in tumorigenesis for over a decade, therapeutic targeting of this pathway has been a challenge. Recently, amlexanox and CYT387, which are in clinical trials for Type II diabetes and myeloproliferative disorders respectively, were identified as potential IKBKE inhibitors. In this study, we uncovered specific novel mechanisms of activation of IKBKE in tumor cells and the outcomes of targeting IKBKE pathway.
Oncogenic mutations are a cause of several human malignancies. Mutations in EGFR are observed in 15% of non-small cell lung cancer patients. While cells expressing these mutations respond better to the first generation TKIs, patients become resistant to these inhibitors due to secondary mutations in EGFR. These mutations were shown to make EGFR constitutively active even in the absence of ligands. Direct targeting of EGFR with secondary mutations has been challenging as EGFR acquires novel mutations upon inhibitor treatment, which confer resistance to the EGFR-TKIs. Hence, it is important to improve our knowledge of the downstream signaling pathways of EGFR. Although PI3K, MEK signaling are well established, mutant EGFR was shown to activate several novel signaling pathways such as miRNA processing and autophagy that are implicated in resistance to EGFR-TKIs.
Here, we show that IKBKE acts downstream of mutant EGFR to activate the NFκB and AKT pathways. In addition, we show that mutant EGFR but not wildtype EGFR can directly phosphorylate IKBKE at Tyrosine 153 and Tyrosine 179 residues that are important for activation of IKBKE kinase. We also found that the IKBKE/TBK1 inhibitor Amlexanox exhibits increased efficacy in inhibiting cell viability in NSCLC cells with activating EGFR mutations. Furthermore, we also found that IKBKE inhibitors activate the MAPK pathway, and EGFR-TKI resistant NSCLCs exhibit enhanced response to co-treatment with IKBKE inhibitors and MEK inhibitors.
Similar to lung cancer, pancreatic cancer is a challenging disease due to lack of direct inhibitors of the KRas mutations that are observed in more than 95% of pancreatic cancer patients. IKBKE/TBK1 pathway is important for KRas signaling, but the efficacy of IKBKE inhibitors in pancreatic cancers is not well studied. Here, we show that IKBKE is an important target in pancreatic cancers that regulates pancreatic cell viability, cell migration and cancer stem cells. Importantly, we provide mechanistic insights into the effects of IKBKE inhibitors on specific signaling pathways. We found that IKBKE inhibition results in significantly increased expression of RTKs, such as ErbB3 and IGF1-R, which increases ERK1/2 activation. Our findings provide support for novel combination strategies for pancreatic cancer.
Metastasis is a poor prognostic factor for ovarian cancer. Although patients with early stage ovarian cancer with no distal metastasis exhibit a 70% 5-year survival rate, Stage IV patients with distal metastasis exhibit only 20% 5-year survival rate. Hence, ongoing efforts are aimed at targeting the pathways that regulate metastasis in ovarian cancers. IKBKE is upregulated in ovarian cancer patients, and IKBKE expression is known to regulate the expression of several genes important for cell motility in ovarian cancers. IKBKE is also implicated in chemo-resistance in ovarian cancer, and siRNA knockdown of IKBKE increases sensitivity towards chemotherapy. However, the mechanistic role of IKBKE in chemo-resistance in ovarian cancer is not known. EphA2 is another well studied oncogene in ovarian cancer as 70% of ovarian cancer patients exhibit elevated levels of EphA2. By activating Focal Adhesion Kinases (FAK), EphA2 can induce metastasis in ovarian cancers. In this study, we show that the clinical PARP inhibitor Olaparib (AZD2281) activates IKBKE by EphA2-mediated tyrosine phosphorylation. We also found that phosphorylation of EphA2 or IKBKE expression can be used as a biomarker for olaparib resistance.
Together, these studies have shed light on novel mechanisms of regulation of IKBKE and their importance in therapy resistance. These observations form a strong pre-clinical proof-of-concept to study the inhibitors further in the clinic.
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Subedee, Ashim. "Molecular Determinants and Transcriptional Regulators in Triple Negative Breast Cancer." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:23845415.
Full textAbstract:
Breast cancer is a highly heterogeneous disease with differences in histopathological and biological characteristics, variable prognoses, and response to therapy. Clinically, breast
tumors are classified based on the expression of hormone receptors (ER and PR) and HER2 as hormone receptor positive (ER+, PR+), HER2+, and triple negative (ER-, PR-, HER2-). Based
on gene expression profiling, breast cancers have been classified into luminal (luminal A and B), HER2+, basal-like and claudin-low subtypes. Knowledge of the molecular properties of luminal and HER2+ subtypes has led to the development of endocrine and HER2-targeted therapies. However, the molecular determinants and transcriptional regulators of basal-like tumors that constitute the majority of triple negative breast cancer (TNBC) are poorly understood. In this dissertation, we have defined some of the molecular characteristics of the basal-like breast cancer phenotype and also identified multiple transcriptional regulators specific to TNBCs.
By using three different reprogramming approaches – somatic cell fusion, nuclear
reprogramming, and transcription factor transduction, we showed that the basal-like breast cancer phenotype is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. We found that luminal breast cancers share a common core epigenetic program, whereas basal-like breast cancers are highly heterogeneous. We demonstrated that protein extracts of basal-like breast cancer cells can reprogram a subset of
luminal breast cancer cells to a basal-like state. Additionally, we identified three transcription factors, EN1, TBX18, and TCF4, the overexpression of which induced the repression of some
luminal features in luminal breast cancer cells.
We also performed a targeted cellular viability screen for selected transcription factors differentially expressed between triple negative and other breast cancer subtypes and identified multiple factors essential for TNBCs including EN1 and TRIP13. We found that downregulation of EN1 and TRIP13 preferentially and significantly reduce cellular viability, colony formation, and in vivo tumorigenicity of TNBC cell lines. We demonstrated that downregulation of EN1 induces an arrest in the G1 phase of the cell cycle and apoptosis. By analyzing the gene expression and histone H3 lysine 27 acetylation (H3K27ac) profiles of TNBC cell lines following downregulation of EN1, we found that EN1 regulates genes involved in angiogenesis, neurogenesis, cell matrix interactions, and WNT signaling pathways. We also performed ChIP-seq for exogenously expressed HA-tagged EN1 to identify its genomic targets. Lastly, we showed that the expression of EN1 correlates with shorter overall survival among patients with basal-like breast tumors. Similarly, by analyzing the gene expression profiles of TNBC cell lines following downregulation of TRIP13, we found that TRIP13 regulates genes involved in IL6 signaling, cell proliferation, and angiogenesis; in line with this we confirmed reduced levels of JAK2 and phospho-STAT3 following TRIP13 downregulation.
In summary, we have unraveled some of the molecular mechanisms of basal-like and luminal breast cancer cell phenotypes and identified factors that might repress luminal differentiation programs in basal-like breast tumors. We have also identified multiple triple negative breast cancer specific transcription regulators. We believe these studies have increased our molecular understanding of basal-like and triple negative breast cancers and have provided potential therapeutic targets for these breast tumors.Medical Sciences
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Labonte, Laura E. "Vascular progenitor cell and extracellular matrix protein interactions in cancer." Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28878.
Full textAbstract:
Vascular progenitor cells (VPCs) facilitate angiogenesis and vascular repair in damaged tissues where inflammatory cytokines coordinate healing through VPC mobilization. VPCs also facilitate tumor angiogenesis and induce angiogenic switching in metastatic sites.
VPC binding to extracellular matrix proteins enriched in injured healing tissues and metastatic sites and the participation of integrins in this binding was investigated. VPC binding profiles on fibronectin and laminin of "healthy" controls was different from those of "cancer" patients and patients with acute tissue damage. Specific integrin-mediated binding of cells in VPC clusters were matrix protein-dependent. Patients with oral cancer and lymphoma had elevated plasma vascular endothelial growth factor (VEGF) levels compared to healthy controls. VEGF facilitated VPC clustering on fibronectin and incubating cells from healthy controls with VEGF switched binding profiles from "healthy" to "cancer". This study provided new insights regarding VPC-matrix interactions that help distinguish VPC involvement in vascular repair from vascular-mediated metastasis.
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Hosseini, Shirazi Seyed Farshad. "Cell cycle dependency of cisplatin cytotoxicity on ovarian cancer cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0028/NQ36776.pdf.
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Leung, Yu Hing Nelly. "Carcinoembryonic antigen cell adhesion molecule 1: cancer and metabolic regulation." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=18740.
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Carcinoembryonic antigen cell adhesion molecule 1, CEACAM1, is a glycoprotein and a member of the CEA (carcinoembryonic antigen) family of genes. It is expressed on the surface of epithelial, endothelial, and hematopoietic cells, within the gastrointestinal tract, reproductive organs, liver, lungs and kidney. CEACAM1 plays a role in inhibiting the immune response, in cell adhesion, in insulin clearance, in cellular apoptosis and proliferation, in angiogenesis, and functions as a tumor suppressor. In humans, CEACAM1 is dysregulated and often lower in hyperplastic lesions than in normal tissue, especially in cases of colon, prostate, liver, and 30% of breast cancers. The role of CEACAM1 in tumor formation was studied using the Ceacam1-/- mouse model, which sustained systemic ablation of CEACAM1. In the absence of CEACAM1, epithelial cells from small intestine and colon undergo less apoptosis than in wild-type cells. Moreover, there is increased proliferation in the Ceacam1-/- colonocytes. As CEACAM1 inactivation alone is not sufficient to induce tumors in the mice, the chemical carcinogen, azoxymethane, was used to induce colon tumors. Ceacam1-/- mice developed a higher tumor burden than wild-type mice. One drawback to chemical carcinogenesis is that multiple mutations in unidentified genes cause tumors. To understand the contribution of CEACAM1 to tumor development, the Ceacam1-/- mice was mated with the genetically modified mouse Apc1638N/+ that spontaneously forms small intestinal tumors. Compound Apc1638N/+: Ceacam1-/- mice developed more tumors than Apc1638N/+ mice, and these tumors progressed to a more advanced stage. In addition, Ceacam1-/- enterocytes showed compromised Wnt signalling. The Ceacam1-/- mouse is also a model for obesity and CEACAM1 is a key factor in insulin clearance. Due to defective insulin clearance, the Ceacam1-/- mice suffer from insulin resistance, and altered lipid synthesis. As these mice age, their weight and abdominalLa glycoprotéine CEACAM1 (Carcinoembryonic antigen cell adhesion molecule 1) est une molécule d'adhésion appartenant à la famille des CEA (Antigène carcinoembryonnaire). CEACAM1 se retrouve à la surface des cellules épithéliales, endothéliales, et hématopoétiques, du tube digestif, des organes reproducteurs, du foie, des poumons et des reins. CEACAM1 présente plusieurs fonctions: inhibition de la réponse immunitaire, adhésion cellulaire, clairance de l'insuline, apoptose et prolifération cellulaire, angiogénèse, et rôle de suppresseur de tumeurs. L'expression de CEACAM1 est souvent diminuée dans plusieurs cas de cancers humains dont le côlon, le foie, la prostate, et 30% des cancers du sein. Le modèle de souris Ceacam1-/- a été utilisé afin d'étudier le rôle de CEACAM1 dans la formation de tumeur. En l'absence de CEACAM1, le tissu épithélial de l'intestin grêle et du côlon subit moins d'apoptose. En contrepartie, une augmentation de la prolifération est détectée dans le côlon des souris Ceacam1-/-. L'absence de CEACAM1 n'étant pas suffisante pour induire l'apparition de tumeurs, nous avons utilisé un cancérogène chimique, l'azoxymethane. Les tumeurs observées dans le côlon des souris Ceacam1-/- s'avèrent être plus importantes que celles des souris contrôles. Le désavantage de l'utilisation de l'azoxymethane est que celui-ci provoque des mutations dans plusieurs gènes plus ou moins définis. Afin de comprendre la contribution de CEACAM1 dans le développement tumoral, la souris Ceacam1-/- a été croisée avec la souris Apc1638N/+ qui forme spontanément des tumeurs dans l'intestin grêle. Les souris Apc1638N/+: Ceacam1-/- développent plus de tumeurs présentant un phénotype tumoral plus agressif que celui formé dans les souris Apc1638N/+. Par ailleurs, les cellules épithéliales de l'intestin grêle Ceacam1-/- montrent une dérégulation de la voie de signalisation Wnt. CEACAM1 étant un facteu
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Austin, Lauren Anne. "Exploring some aspects of cancer cell biology with plasmonic nanoparticles." Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/54236.
Full textAbstract:
Plasmonic nanoparticles, specifically gold and silver nanoparticles, exhibit unique optical, physical, and chemical properties that are exploited in many biomedical applications. Due to their nanometer size, facile surface functionalization and enhanced optical performance, gold and silver nanoparticles can be used to investigate cellular biology. The work herein highlights a new methodology that has exploited these remarkable properties in order to probe various aspect of cancer cell biology, such as cell cycle progression, drug delivery, and cell death. Cell death mechanisms due to localized gold and silver nanoparticle exposure were also elucidated in this work. Chapter 1 introduces the reader to the synthesis and functionalization of gold and silver nanoparticles as well as reviews their implementation in biodiagnostic and therapeutic applications to provide a foundation for Chapters 3 and 4, where their use in spectroscopic and cytotoxic studies are presented. Chapter 2 provides the reader with detailed explanations of experimental protocols for nanoparticle synthesis and functionalization, in vitro cellular biology experiments, and live-cell Raman spectroscopy experiments that were utilized throughout Chapters 3 and 4. Chapter 3 presents the use of nuclear-targeted gold nanoparticles in conjunction with a Raman microscope modified to contain a live-cell imaging chamber to probe cancer cell cycle progression (Chapter 3.1), examine drug efficacy (Chapter 3.2), monitor drug delivery (Chapter 3.3), and detect apoptotic molecular events in real-time (Chapter 3.4). In Chapter 4, the intracellular effects of gold and silver nanoparticles are explored through live-cell Rayleigh imaging, cell cycle analysis and DNA damage (Chapter 4.1), as well as through the elucidation of cytotoxic cell death mechanisms after nanoparticle exposure (Chapter 4.2) and live cell imaging of silver nanoparticle treated cancer cell communities (Chapter 4.3).
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Avva, Jayant. "Complex Systems Biology of Mammalian Cell Cycle Signaling in Cancer." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1295625781.
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Dive, C. "Flow cytoenzymology with special reference to cancer chemotherapy." Thesis, Open University, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.384585.
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Cerone, Maria Antonietta. "Telomere maintenance in human cells : implications in cancer and ageing diseases." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86067.
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Telomeres are protective structures at the end of eukaryotic chromosomes essential for indefinite cell proliferation. Their disruption causes activation of DNA repair pathways, growth arrest and/or cell death. In normal cells telomere shortening during cell division has been proposed to act as a tumor suppressor mechanism to block the proliferation of cells at risk of undergoing malignant transformation. Overcoming this proliferative block by activating a mechanism to maintain telomeres is a necessary requirement for unlimited proliferation and tumor progression. Human cells have two mechanisms for telomere maintenance: a more common one based on telomerase and a rarer one based on recombination called ALT.Here we report the isolation of an immortal human cell line that maintains short telomeres in the absence of biologically active telomerase and key features of active ALT. Our results suggest that the mechanisms of telomere maintenance in human cells may be more diverse than previously thought and have important implications for the development of anti-cancer strategies based on the inhibition of telomere maintenance.
Due to widespread distribution of telomerase in human tumors and its absence in most normal cells, telomerase is the main target of these anti-cancer strategies. However, targeting telomerase per se or in combination with anti-cancer drugs is not sufficient to trigger rapid cell death of tumor cells. On the other hand, disturbances in telomere capping do not require telomere shortening to induce growth arrest and may act more quickly. Our goal was to investigate the feasibility of a new approach based on the combination of telomere destabilization and chemotherapeutic drugs. Our results show that interfering with telomere maintenance enhances the susceptibility of human tumor cells to anti-cancer drugs independently of their telomere lengths and mechanisms to maintain them.
Finally, given the involvement of telomeres in maintaining genomic stability, we investigated the mechanism by which mutations in the telomerase RNA subunit contribute to autosomal dominant dyskeratosis congenita, a premature ageing disease associated with mutations in the telomerase holoenzyme. Our data strongly indicate that the clinical manifestations of this disease may be caused by telomere shortening due to haploinsufficiency of telomerase activity and provide a direct correlation between disturbances in telomere length maintenance and human disease.
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Sarkar, Abby Joya. "Investigating the Role of Sox2 in Stomach Tissue Homeostasis and Cancer." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467240.
Full textAbstract:
The transcription factor Sox2 is essential for the establishment and maintenance of multiple stem cell populations and its coding region is amplified in certain carcinomas. However, Sox2’s role in stomach homeostasis and cancer is poorly understood. In this thesis, I used mouse genetics to investigate the expression pattern and function of Sox2 in the adult stomach during normal tissue homeostasis and tumorigenesis. Using a genetic lineage tracing system, I found that Sox2 expression marks a gastric stem cell population capable of self renewal and differentiation throughout the lifetime of a mouse, raising the key question of whether Sox2 itself is required for adult stomach function. Using a combination of novel mouse models, I examined the consequences of Sox2 loss of function on stomach regeneration as well as the susceptibility of Sox2+ cells to transformation. Surprisingly, I found that Sox2 itself is dispensable during stomach homeostasis, although Sox2-expressing cells readily give rise to Wnt-driven adenomas. To gain insight into the molecular function of Sox2, I performed ChIP-Seq analysis which revealed that
the majority of Sox2 targets in mouse gastric stem and progenitor cells are related to tissue-specific functions such as endoderm development, Wnt signaling and gastric cancer while only a small set of genes overlaps with targets occupied by Sox2 in other stem cell populations.
SOX2 has been described as an amplified oncogene in several types of human cancers derived from the foregut endoderm including lung and esophageal squamous cell carcinomas. Unexpectedly, I found that Sox2 loss enhances stomach tumor formation and organoid growth in an Apc/Wnt depdendent adenoma mouse model. Using a reporter assay, I further showed that altered Sox2 levels modulate
Tcf/Lef-dependent transcription, providing a molecular explanation for the observed proliferation phenotypes. In summary, my genetic and molecular studies offer insight into how Sox2 regulates stomach tissue homeostasis and cancer and evidence that Sox2’s mode of action is context and tissue
specific.Medical Sciences
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Hwang, Katie Lee. "The Metabolic Role of the Hippo Pathway in Liver Development and Cancer." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467350.
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Hepatocellular carcinoma (HCC) is a global health problem with poor prognosis and limited therapeutic options. While the clinical risk factors for HCC are well described, the precise molecular and metabolic mechanisms contributing to malignant transformation remain largely unknown. Recently, the Hippo signaling pathway has been identified as a key regulator of cellular proliferation, organ size, and tumorigenesis in numerous tissues, including the liver. However, the metabolic impact of the pathway in supporting liver growth and tumorigenesis has not been studied. The zebrafish, Danio rerio, has successfully been applied as a model to investigate signaling pathways important in organ development to model liver development and cancer. Here, we utilize the zebrafish to investigate the functional and metabolic roles of the Hippo pathway in liver development and cancer in vivo.
Using a transgenic zebrafish model with liver-specific activation of the transcriptional co-activator Yap, the downstream target of the Hippo pathway, we show Yap is functionally conserved in its ability to promote embryonic and adult hepatomegaly. These livers demonstrate signs of dysplasia and increased tumor susceptibility upon chemical carcinogen exposure. Using transcriptomic and metabolomic analysis, we discover that nitrogen metabolism is significantly altered in Yap-transgenic livers. Yap upregulates glutamine synthetase (Glul) expression leading to elevated steady-state levels of glutamine, which significantly contributes to its ability to enhance liver growth and de novo purine biosynthesis.
To further probe the functional and metabolic role of Yap prior to liver outgrowth, we utilize yap knockout zebrafish and heat-shock inducible transgenic zebrafish that modulate
Yap activity to examine early liver development. We show Yap is important for hepatoblast formation and expansion. Further, Yap modulates glucose uptake and glycolytic flux into de novo nucleotide synthesis. Overall, this dissertation reveals novel roles of Yap in cellular metabolism to support proliferation and growth by directing glucose into the building blocks of DNA in the context of development and cancer.Medical Sciences
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Schlueter, Daniela K. "A multiscale systems biology study of in vitro cell migration and cancer cell invasion." Thesis, University of Dundee, 2013. https://discovery.dundee.ac.uk/en/studentTheses/9e7f0415-f565-4a3d-ade3-f3eb540bf088.
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German, Natalie Janelle. "Investigating and Exploiting Metabolic Vulnerabilities in Cancer." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467175.
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Fuel metabolism in healthy cells is not sufficient to sustain the biosynthetic and energetic demands of cancer. For a normal cell to transform to a rapidly dividing tumor cell, metabolism must be dramatically altered in a process called metabolic reprogramming, characterized by increased nutrient uptake and re-purposing. As we move toward a future of personalized medicine, there is new opportunity in targeting the metabolic requirements specific to an individual’s tumor. To this end, it is critical to understand molecular drivers that cancer cells hijack to modify metabolism.
In this dissertation, I describe three studies on enzymes and metabolic pathways that shed light on molecular regulation of metabolic reprogramming in cancer. First, we screened for substrates of SIRT4, a mitochondrial sirtuin that promotes metabolic homeostasis and suppresses cancer by mechanisms not well understood. We used proteomics to identify hyperacetylated mitochondrial proteins in SIRT4 knockout mouse tissues compared to wildtype. We find SIRT4 binds and inhibits pyruvate carboxylase, an enzyme important for refueling the TCA cycle in cancer, indicating SIRT4 may target this node in tumor metabolism. Second, we reveal a role for prolyl hyrdoxylase domain (PHD) 3 in coordinating cancer cell addition to fat catabolism. In biochemical and cellular studies, we find PHD3 hydroxylates and activates acetyl-CoA carboxylase (ACC2) to repress fatty acid oxidation (FAO). Loss of this regulatory axis in leukemia enables greater utilization of fatty acids as fuel, and also serves as a liability by rendering cells susceptible to FAO inhibition. Finally, we used metabolomics to define alterations caused by the diabetes drugs metformin and phenformin to better understand their anti-cancer properties. We analyzed the drugs’ effects on cells undergoing neoplastic transformation and on cancer stem cells (CSCs), a small population that possesses predominant tumor-initiation capacity and is selectively inhibited by metformin. We show metformin and phenformin induce changes that oppose cancer cell survival by eliciting a nutrient crisis during transformation and depleting nucleotide triphosphates in CSCs. In sum, these findings contribute to the future potential to impede nutrient switches in cancer, thus turning the metabolic dependencies of cancer cells into metabolic vulnerabilities.Medical Sciences
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Hoopes, Justin Darrel. "Mechanisms of Induced Cell Death in Bluetongue Virus Challenged Human Cell Lines." DigitalCommons@USU, 2009. https://digitalcommons.usu.edu/etd/252.
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Bluetongue virus (BTV) is a pathogenic member of the Reoviridae family. BTV does not cause disease in humans, but is capable of selectively infecting and killing certain transformed human cell lines. Understanding BTV's oncotrophism may lead to new therapeutics for treating cancer. This study focused on the underlying mechanisms of BTV-induced cell death in carcinoma cell lines. It was our hypothesis that BTV infects human carcinoma transformed cells, produces mRNA and protein, induces a strong inflammatory response, induces mitogen activated protein kinase (MAPK)-based pro-apoptotic signaling, inhibits PKB-based signaling, and eventually kills the cell by inducing apoptosis.
Three carcinoma cell lines (A498, HEP-G2, and A549) were independently infected with BTV. In each cell line we determined: (1) cell viability over the course of infection; (2) BTV induced cytokine expression profile and magnitude of expression; (3) BTV viral RNA expression profile and magnitude of expression; (4) BTV viral protein expression profile and magnitude of expression; (5) changes in BTV induced cell death and cytokine expression in cells with protein kinase B (PKB), p38-MAPK, extracellular receptor kinase (ERK-1/2), stress-activated protein kinase (SAPK-JNK), Src kinase, platelet-derived growth factor receptor (PDGFR) kinase, epidermal growth factor receptor (EDGFR) kinase, or Janus kinase (JAK) activity inhibited; (6) intracellular changes in PKB, p38-MAPK, ERK-1/2, and SAPK-JNK phosphorylation as a result of BTV infection; and (7) BTV-induced changes in tyrosine phosphorylation.
We determined that BTV infects and kills all three cell lines in a cell line dependent manner. Relative cell death between cell lines was proportional to cytokine expression, but inversely proportional to viral protein expression. Only tyrosine kinase inhibitors influenced BTV-induced cell death and cytokine expression. Both A498 and A549 cells constitutively expressed phosphorylated PKB and p38 MAPK, of which both were de-phosphorylated during BTV infection. Tyrosine phosphorylation remained active, with elevated tyrosine phosphorylation exclusively in infected cells.
We conclude that BTV-induced cell death and cytokine expression are a function of the cell's response to infection and are directly related through intracellular signaling. These pathways are only partially poly I:C inducible, but include PKB and tyrosine kinase signaling.
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Chinaranagari, Swathi. "Epigenetic Silencing of ID4 in Prostate Cancer: Mechanistic Insight." DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 2015. http://digitalcommons.auctr.edu/cauetds/13.
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Inhibitor of DNA binding/differentiation protein 4 (ID4) is a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. ID4 shares the homology of HLH domain with other ID proteins (ID1, ID2, and ID3) and lack the basic DNA binding region. Evidence suggested that unlike ID1, ID2 and ID3, ID4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promoting apoptosis. Consistent with these observations ID4 is epigenetically silenced in DU145 prostate cancer cell line. In this study we investigated whether ID4 is also epigenetically silenced in prostate cancer. We also examined association between ID4 promoter hyper-methylation and its expression in prostate cancer cell lines. ID4 protein expression was analyzed in human prostate adenocarcinoma samples by Immunohistochemistry (IHC). ID4 promoter methylation pattern on prostate cancer cell lines was examined by methylation specific PCR. In addition, we performed methylation specific PCR on the human prostate tissues and genomic DNA to correlate cell line studies with clinical studies. IHC demonstrated decreased ID4 protein expression in human prostate tissue samples, whereas higher nuclear ID4 expression was found in normal prostate tissues. ID4 methylation specific PCR (MSP) on prostate cancer cell lines, showed ID4 methylation in DU145, but not in LNCaP and C33 cells. C81 and PC3 cells showed partial methylation. Increased ID4 methylation in C81 as compared to LNCaP suggests its epigenetic silencing as cells acquire androgen independence. Tumors with ID4 promoter hyper-methylation showed distinct loss of ID4 expression. However, the underlying mechanism involved in epigenetic silencing of ID4 is currently unknown. We hypothesized that ID4 promoter methylation is initiated by an EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27Me3). ID4 expressing (LNCaP) and non-expressing (DU145 and C81) prostate cancer cell lines were used to investigate EZH2, H3K27Me3 and DNMT1 enrichment on ID4 promoter by Chromatin immuno-precipitation (ChIP). Increased enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and C81 cell lines was compared to ID4 expressing LNCaP cell line. Knockdown of EZH2 in DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent manner. ChIP on prostate cancer tissue specimens and cell lines suggested EZH2 occupancy and H3K27Me3 marks on the ID4 promoter. Collectively, our data indicate a PRC2 dependent mechanism in ID4 promoter silencing in prostate cancer through recruitment of EZH2 and a corresponding increase in H3K27Me3. Increased EZH2, but decreased ID4 expression in prostate cancer strongly supports this model.
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Eng, Jamei Raena. "Localization of anthracyclines in drug resistant human MCF-7 breast cancer cells." Thesis, University of Ottawa (Canada), 2007. http://hdl.handle.net/10393/27841.
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Multidrug resistance (MDR) commonly occurs during the treatment of cancer. Current research has focused mostly on the role of drug transporters, as the main mechanism of MDR; however, few have demonstrated a definite link between the expression or function of drug transporters and MDR in cancer patients.
Anthracyclines such as doxorubicin and epirubicin, autofluoresce and can be monitored by confocal microscopy. Two of the four resistant cell fines generated in our lab: the MCF-7EPI cells and to some extent MCF-7 DOX cells, exhibit a localization defect, whereby epirubicin is localized primarily in the cytoplasm rather than the nucleus. This drug localization defect temporally correlated with the onset of drug-resistance during selection for drug resistance in these cell lines. Consistent with the possible sequestration of drugs into acidic vesicles, acridine orange staining has revealed the presence of aggregates of acidified vesicles in the perinuclear region of MCF-7EPI cells. However, co-localization experiments using a number of intracellular organelle markers determined that epirubicin was localized to lysosomes and not consistently to acidic vesicles. An inhibitor of vacuolar H+ ATPase, was unable to restore the localization of epirubicin to the nucleus. Immunofluorescence using an ABCB1 antibody revealed the localization of ABCB1 predominantly in the plasma membrane and to some extent in the perinuclear region of MCF-7EPI cells. Nevertheless, inhibitors of this transporter failed to restore localization of epirubicin to the nucleus. Taken together, these findings strongly suggest that the acquisition of epirubicin resistance in breast tumour cells may involve the P-glycoprotein independent sequestration of drug into lysosomes. These lysosomes need not be acidic, nor does the removal of acid vesicles by inhibition of vacuolar H+ ATPases block the sequestration of drug into lysosomes.
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Gerrard, Diana Lea. "Characterization Of Epigenetic Plasticity And Chromatin Dynamics In Cancer Cell Models." ScholarWorks @ UVM, 2019. https://scholarworks.uvm.edu/graddis/1060.
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Cancer progression is driven by cumulative changes that promote and maintain the malignant phenotype. Epigenetic alterations are central to malignant transformation and to the development of therapy resistance. Changes in DNA methylation, histone acetylation and methylation, noncoding RNA expression and higher-order chromatin structures are epigenetic features of cancer, which are independent of changes in the DNA sequence. Despite the knowledge that these epigenetic alterations disrupt essential pathways that protect cells from uncontrolled growth, how these modifications collectively coordinate cancer gene expression programs remains poorly understood. In this dissertation, I utilize molecular and informatic approaches to define and characterize the genome-wide epigenetic patterns of two important human cancer cell models. I further explore the dynamic alterations of chromatin structure and its interplay with gene regulation in response to therapeutic agents.
In the first part of this dissertation, pancreatic ductal adenocarcinoma (PDAC) cell models were used to characterize genome-wide patterns of chromatin structure. The effects of histone acetyltransferase (HAT) inhibitors on chromatin structure patterns were investigated to understand how these potential therapeutics influence the epigenome and gene regulation. Accordingly, HAT inhibitors globally target histone modifications and also impacted specific gene pathways and regulatory domains such as super-enhancers. Overall, the results from this study uncover potential roles for specific epigenomic domains in PDAC cells and demonstrate epigenomic plasticity to HAT inhibitors.
In the second part of this dissertation, I investigate the dynamic changes of chromatin structure in response to estrogen signaling over a time-course using Estrogen Receptor (ER) positive breast cancer cell models. Accordingly, I generated genome-wide chromatin contact maps, ER, CTCF and regulatory histone modification profiles and compared and integrated these profiles to determine the temporal patterns of regulatory chromatin compartments. The results reveal that the majority of alterations occur in regions that correspond to active chromatin states, and that dynamic chromatin is linked to genes associated with specific cancer growth and metabolic signaling pathways. To distinguish ER-regulated processes in tamoxifen-sensitive and in tamoxifen-resistant (TAMR) cell models, we determined the corresponding chromatin and gene expression profiles using ER-positive TAMR cancer cell derivatives. Comparison of the patterns revealed characteristic features of estrogen responsiveness and show a global reprogramming of chromatin structure in breast cancer cells with acquired tamoxifen resistance.
Taken together, this dissertation reveals novel insight into dynamic epigenomic alterations that occur with extrinsic stimuli and provides insight into mechanisms underlying the therapeutic responses in cancer cells.
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Henry, Whitney Ingrid. "Investigating the Phosphoinositide 3-Kinase (PI3K) Pathway for Therapeutic Strategies for Breast Cancer." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718731.
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Despite major advances in our understanding of the etiology of breast cancer, it remains a leading cause of cancer death in women worldwide. This warrants the search for novel alternatives for diagnosis and therapeutic intervention. The phosphoinositide 3-kinase (PI3K) pathway regulates all aspects of breast cancer development, from initiation to metastatic dissemination. This is underscored by the high prevalence of somatic mutations in PIK3CA, the gene encoding the catalytic subunit of PI3K, in breast cancer. Although targeting the PI3K pathway is a viable therapeutic approach, many PI3K pathway inhibitors have yet to show significant clinical efficacy.
Here we examine the potential chemotherapeutic benefit of aspirin in PI3K-driven breast cancer. We demonstrate that mutant PIK3CA breast cancer cells show a dose-dependent decrease in cell viability and anchorage-independent growth in soft agar, when treated with increasing concentrations of aspirin/salicylate. Co-treatment of aspirin sensitizes mutant PIK3CA breast cancer cells to PI3K inhibitors to enhance suppression of proliferation. Additionally, immortalized mammary epithelial cells expressing mutant PIK3CA H1047R show greater sensitivity to aspirin when compared to cells expressing wild-type PIK3CA grown in three-dimensional (3D) Matrigel culture. Mechanistic studies indicate that the growth inhibitory effect of aspirin are due to activation of AMP-activated protein kinase (AMPK), inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling and induction of autophagy. Importantly, this growth suppression is independent of the activities of cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB).
We have also investigated the transcriptional responses to PI3K activation in breast cancer cells. By performing microarray and gene ontology analysis, we show that constitutive PI3K pathway activation preferentially affects the gene expression of cytokines, chemokines and other secreted factors. We identify a novel PI3K-regulated gene termed long Pentraxin 3 (PTX3), whose functional role in cancer is largely unknown. We demonstrate that the PI3K pathway regulates the expression of PTX3 in an NF-κB dependent manner. Using functional cell-based assays, we demonstrate a role for PTX3 in PI3K-driven proliferation and survival in two-dimensional (2D) monolayer and three-dimensional (3D) Matrigel culture.
Additionally, we identify a novel large intergenic noncoding RNA (lincRNA) transcript termed c14orf34, which is upregulated in both mutant PIK3CA and Src-transformed MCF10A cells. By performing gene expression profiling, we show that this transcript is associated with basal-like breast cancer cells and genomically altered in a subset of invasive breast carcinomas from human patients. By employing ChIP-seq analysis, we demonstrate that c14orf34 is regulated by the transcription factor STAT3. Loss-of-function studies suggest a role for this lincRNA in cell invasion and migration. We also identify several genes that are differentially expressed upon overexpression of c14orf34 in MCF10A cells, indicative of a potential role in regulating global gene expression.Medical Sciences
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MacDonald, Patricia. "Defining functional domains within GPNMB important for breast cancer cell invasion." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96829.
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Glycoprotein non-metastatic melanoma protein B (GPNMB)/Osteoactivin (OA) is a transmembrane protein that is commonly expressed in basal/triple-negative breast cancer. Our group discovered that GPNMB/OA is sufficient to enhance the migration and invasion of weakly metastatic murine breast cancer cells in vitro, and promotes the formation of bone and lung metastases in vivo. In this study, we sought to fully characterize the pro-invasive role of GPNMB/OA in human breast cancer cells by defining those motifs/domains within GPNMB/OA that are required to promote breast cancer cell invasion. In addition, we have sought the identity of putative GPNMB/OA interacting proteins that may be involved in promoting GPNMB/OA-mediated effects on breast cancer cell invasion and metastasis. To accomplish this, a panel of GPNMB/OA mutants were generated and expressed in the GPNMB/OA null BT-549 and MDA-MB-453 human breast cancer cells and subjected to in vitro invasion assays characterization. A candidate approach based on previous literature reports was used to facilitate the identification of GPNMB/OA protein binding partners in addition to attempting mass spectrometry analysis. Finally, a transgenic mouse model was created that expresses human GPNMB/OA under the control of the Mouse Mammary Tumor Virus (MMTV) promoter to further explore the role of GPNMB/OA on mammary gland development and tumorigenesis in vivo. Our results indicate that human GPNMB/OA is sufficient to induce enhanced invasion of BT549 breast cancer cells in vitro, which requires both the cytoplasmic tail and RGD recognition motif. Characterization of the MMTV-GPNMB/OA transgenic mice revealed that GPNMB/OA does not negatively affect normal mammary duct development in virgin females and no mammary tumors have developed to date. We conclude that GPNMB/OA is indeed capable of inducing invasion of breast cancer cells in vitro, which may require the participation of integrins and/or the residues/motifs within the cytoplasmic tail responsible for recruiting signalling molecules to this region of GPNMB/OA.Glycoprotein non-metastatic melanoma protein B (GPNMB), aussi connu sous le nom de Ostéoactivine (OA), est une protéine transmembranaire fréquemment exprimée dans les tumeurs mammaires appartement au sous-type triple négatif. Notre groupe a mis en évidence que l'expression de GPNMB/OA est suffisante pour accroître, in vitro, les capacités migratoires et invasives associées aux cellules murines de cancer du sein faiblement métastatiques. In vivo, l'expression de GPNMB/OA est caractérisée par une augmentation de la formation des métastases osseuses et pulmonaires. Dans cette étude, nous avons cherché à caractériser le rôle pro-invasif associé à l'expression de GPNMB/OA dans les cellules humaines de cancer du sein en identifiant les domaines ou motifs de GPNMB/OA impliqués dans le phénotype invasif des cellules de cancer du sein. D'une part, nous avons entrepris l'identification de protéines interagissant avec GPNMB/OA et qui pourraient participer aux phénotypes associés à l'expression de GPNMB/OA. Pour ce faire, nous avons généré une série de mutants pour la protéine GPNMB et nous les avons exprimées dans les lignées cellulaires BT-549 et MDA-MB-453, qui n'exprime normalement pas GPNMB/OA, que nous avons par la suite soumis à des essais d'invasion in vitro. D'autre part, une étude de la littérature scientifique, associée à une analyse par spectrométrie de masse, ont été utilisées pour identifier les protéines partenaires potentielles de GPNMB/OA. Finalement, nous avons généré une lignée de souris transgénique qui exprime la forme humaine de GPNMB/OA sous le contrôle du promoteur Mouse Mammary Tumor Virus (MMTV). Ce modèle murin a été utilisé pour étudier le rôle de GPNMB/OA sur le développement de la glande mammaire et sur la tumorigenèse in vivo. Ainsi, ces travaux ont démontré que la forme humaine de GPNMB/OA est suffisante pour induire une augmentation des propriétés invasives des cellules BT549 et que ce phénotype requiert à la fois la région cytoplasmique et le motif RGD de la protéine GPNMB/OA. La caractérisation, des souris transgéniques MMTV-GPNMB/OA a révélé, pour sa part, que GPNMB/OA n'interfère pas avec le développement normal des glandes mammaires chez les femelles vierges et aucune tumeur n'a été détectée à ce jour. L'ensemble de nos données démontre que GPNMB/OA est capable d'induire les propriétés invasives associées aux cellules de cancer du sein, et que ce phénotype requiert l'interaction du motif RGD de la protéine GPNMB avec les intégrines et/ou des résidus ou motifs présents dans la partie cytoplasmique de GPNMB/OA et qui pourraient induire le recrutement de molécule de signalisation dans cette région de GPNMB/OA.
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Anderson, Nicole Shree. "The Roles of Elevated Bcl-2 in Ovarian Cancer." Scholar Commons, 2010. http://scholarcommons.usf.edu/etd/6161.
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Ovarian cancer (OC) is the second most common gynecologic cancer; however it is responsible for the most gynecologic cancer-related deaths. Apoptosis evasion is an important mechanism in OC tumorigenesis, and the prototypic anti-apoptotic protein, B-cell lymphoma 2 (Bcl-2), is often overexpressed in OC tumors. Gaining a better understanding of the mechanism(s) behind Bcl-2 overexpression and potential extra-anti-apoptotic functions of Bcl-2 could elucidate the importance of elevated Bcl-2 in OC. In the current study, I show through immunohistochemical analysis of normal, benign, and OC tissue sections, that both epithelial and stromal Bcl-2 expression decreases with OC progression. However, the number of Bcl-2-positive lymphocyte nests and the size of these lymphocyte nests increase dramatically with OC progression. Additionally, this study shows that lysophosphatidic acid (LPA), a glycerophospholipid frequently elevated in serum and ascites fluid of OC patients, upregulates Bcl-2 in OC cells. Bcl-2 enzyme-linked immunosorbant assay (ELISA), western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), and luciferase reporter assays reveal that LPA increases Bcl-2 promoter, messenger RNA (mRNA), and protein levels in OC cells, but not in normal immortalized ovarian surface epithelial (IOSE) cells. LPA also increases secreted levels of Bcl-2. In vitro human umbilical vein endothelial cell (HUVEC) tube formation assays show that OC-derived Bcl-2 or recombinant human (rh) Bcl-2 promotes aberrant formation of tube-like structures. Though extracellular Bcl-2 does not affect HUVEC cell viability, it may cause aberrant tube formation by inhibiting HUVEC migration. Finally, Bcl-2 ELISA reveals that urinary Bcl-2 levels in OC patients are higher than those in normal individuals and patients with benign gynecologic disease. Urinary Bcl-2 also complements serum CA125 when the two are compared in parallel samples. Furthermore, urinary Bcl-2 decreases following cytoreductive surgery. Altogether, the results suggest that Bcl-2 is important in OC tumorigenesis and angiogenesis. Additionally, urinary Bcl-2 may be a valuable non-invasive biomarker for OC diagnosis and/or screening. Consequently, further elucidation of mechanisms of Bcl-2 overexpression and its extra-apoptotic functions could lead to improved treatment and diagnostic strategies for OC patients.
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Li, Xiaodong 1966. "Characterization of the signaling pathways underlying netrin-1 receptor deleted in colorectal cancer." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84281.
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Netrins are a small family of secreted proteins that function as chemotropic cues directing cell and axon migration during neural development. They are bifunctional molecules attracting and repelling different classes of axons. DCC (deleted in colorectal cancer) is a transmembrane receptor for netrin-1 implicated in mediating both responses. The intracellular mechanisms mediating the response of an axon to netrin-1 are currently unclear. Previous studies indicated that extracellular guidance cues induce the neuronal growth cone to advance, retract, or turn by regulating the actin cytoskeleton within the growth cone. The Rho family GTPases, in particular, RhoA, Rac1 and Cdc42, are well-described regulators of actin reorganization in non-neuronal cells, and there is now compelling evidence implicating a role for them as signaling components within the neuronal growth cone.In the first part of this thesis, we have demonstrated that the Rho GTPases are required for embryonic spinal commissural axon outgrowth induced by netrin-1. Using NIE-115 neuroblastoma cells we found that both Rac1 and Cdc42 activities are required for DCC-induced neurite outgrowth. In Swiss 3T3 fibroblasts, DCC was found to trigger actin reorganization through activation of Rac1. These results implicate the small GTPases as important signaling components in the molecular mechanisms underlying DCC.
In the second part, we found that DCC interacts constitutively with the adaptor protein Nck in commissural neurons. Moreover, dominant negative Nck-1 inhibits the ability of DCC to induce neurite outgrowth in NIE-115 cells and to activate Rac1 in fibroblasts in response to netrin-1. These studies provide evidence for an important role of Nck-1 in a novel signaling pathway from an extracellular guidance cue to changes in the actin-based cytoskeleton responsible for axonal guidance.
In the last part, we found that disruption of each of the PxxP motifs in the cytoplasmic domain of DCC is not able to block the interaction of DCC with Nck-1, suggesting that more than one PxxP motifs or non-PxxP sequences may mediate the interaction of DCC with Nck-1.
Taken together, the data in this thesis contribute to our understanding of the intracellular mechanisms mediating the response of an axon to netrin-1 during neural development.
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Xia, Xianfang. "Cell Shape and Treatment Duration: How They Influence a Cancer Cell's Response to TNF." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493296.
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The purpose of my research was to investigate the sources of heterogeneity in cellular decisions that are based on both external and internal cues. I used the signaling network induced by tumor necrosis factor (TNF) as a model system to examine how the duration of a stimulus and cell shape may affects signaling and cellular decisions. First, using a microfluidic device to achieve fine control of the ligand delivery to cells, my colleagues and I found that the duration of TNF stimulation is a factor that coordinates cell fate decisions in single cells. Specifically, we found that a few seconds of exposure to TNF is sufficient to activate the NF-κB pathway and induce apoptotic cell death and that, strikingly, a 1-min pulse of TNF can be more effective at killing cells than a 1-hour pulse. Second, to study the effects of cell shape, I used a two-pronged approach. Initially, I used live-cell imaging and single-molecule fluorescence in situ hybridization (smFISH) to examine whether descriptors of cell shape correlate with NF-κB nuclear translocation and NF-κB-dependent transcription in unperturbed populations of single cells. Next, I used surface micro-patterning to impose different geometry and degrees of spreading on cells and examine NF-κB-dependent transcription in these cells. I found that descriptors that quantify cell spreading, such as cell area and nuclear area, correlate with NF-κB nuclear translocation and NF-κB-dependent transcription. In addition, imposing bigger amount of spreading on cells increased the transcript abundance for two NF-κB-dependent genes, A20 and IκBα. In contrast, the relationships between geometry-related cell shape descriptors and NF- κB-dependent transcription are more subtle and complex. Importantly, despite observing the correlations between cell spreading and NF-κB activity, I found that the effects of cell shape on NF-κB dynamics and on NF-κB-dependent transcription were small. Together, my investigations of TNF-induced signaling have shown that while the duration of treatment encodes information used in the TNF-induced cell death decision, NF-κB dynamics and NF-κB-dependent transcription are quite robust to changes in cell shape.Medical Sciences
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Gao, Chen. "Role of SPDEF in Prostate Cancer." University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1343051932.
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Dai, Meiou. "Regulation of TGFß signaling on tumor cell migration, invasion and stem cell activity in triple negative breast cancer." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119559.
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Basal-like triple negative breast cancers (TNBCs) display poor prognostic features with larger tumor size, higher tumor grade, and an increased risk for lymph node and distant metastasis as well as tumor recurrence. Transforming growth factor beta (TGFβ) is a key regulator of the cellular processes by which breast cancer cells from the primary tumor metastasize to distant organs. However, the molecular mechanisms underlying TGFβ's pro-metastatic effects remain to be fully elucidated. Here, we investigated the role of TGFβ signaling pathway in regulating cell migration, invasion and cancer stem cell self-renewal capacity, which are the initial and critical steps in breast cancer metastasis. Our studies initially identified a novel function for the cell cycle regulator p21 and its binding partner acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-induced TNBC cell migration and invasion in vitro as well as tumor invasiveness in vivo. As p21 can interact with different cyclin and CDK complexes, we investigated whether other cell cycle regulators are also involved in TGFβ-induced tumor progression. We found that TGFβ promotes physical interaction and nuclear co-localization between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins promote tumor growth and locally invasive tumors. In addition, we found that TGFβ can activate cyclin D1/CDK4 complex to promote cancer stem cell activity and self-renewal capacity in TNBC. Together, we have defined p21, cyclin D1 and CDK4 as key downstream regulators of TGFβ tumor-promoting functions.Le cancer du sein triple-négatif (CSTN) de type basal démontre des signes cliniques caractéristiques d'un mauvais pronostique, tels qu'une taille et un grade plus élevés des tumeurs, un risque augmenté de développer des métastases lymphatiques et distantes, ainsi qu'un plus grand risque de récurrence de la maladie. Le TGFβ est un régulateur clé des procédés cellulaires par lesquels les cellules cancéreuses du cancer du sein se détachent de la tumeur primaire pour former des métastases aux organes distants. Cependant, les mécanismes moléculaires par lesquels le TGFβ accomplit son rôle pro-métastatique restent à être élucidés. Ici, nous investiguons le rôle du TGFβ dans la régulation de la migration cellulaire, l'invasion, et la capacité des cellules souches à se renouveler, qui sont les étapes initiales et critiques à la formation de métastases dans le cancer du sein. Notre étude a tout d'abord identifié une nouvelle fonction pour le régulateur de cycle cellulaire p21 et son partenaire associé, l'acetyltransférase pCAF, comme étant des régulateurs de transcription dont la présence est critique pour la migration et l'invasion cellulaire in vitro et l'invasion tumorale in vivo médiées par le TGFβ pour le CSNT. Le p21 pouvant interagir avec différents complexes de cycline et CDK, nous avons investigué si d'autres régulateurs du cycle cellulaire sont aussi impliqués dans la progression tumorale médiée par le TGFβ. Nous avons trouvé que le TGFβ promeut l'interaction physique et la co-localisation nucléaire entre la cycline D21 et p21. La co-expression de la cycline D1 et de p21 promeut la croissance tumorale et l'invasion locale des tumeurs. De plus, nous avons trouvé que le TGFβ peut activer le complexe cycline D1/CDK4 pour promouvoir l'activité des cellules souches cancéreuses, ainsi que leur capacité de renouvèlement dans le CSNT. Ces résultats nous ont permis de définir p21, la cycline D1, et CDK4, comme des régulateurs clés en aval du TGFβ dans ses fonctions pro-métastatiques
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Mercado-Matos, Jose R. "A Mechanistic Investigation of Insulin Receptor Substrate 2 Function in Breast Cancer Progression." eScholarship@UMMS, 2017. http://escholarship.umassmed.edu/gsbs_diss/918.
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The advancement of cancer treatment depends on understanding the biological processes that contribute to disease progression. The spread of tumor cells from the primary site to distant organs is the biggest obstacle to efficacious treatment. The insulin receptor substrate (IRS) proteins IRS1 and IRS2 are cytoplasmic adaptor proteins that organize signaling events downstream of the Insulin receptor (IR) and the Insulin-like growth factor receptor 1 (IGF1R). Both of these receptors have been implicated in cancer progression. The IRS proteins share a significant level of homology and are both capable of recruiting and activating phosphatidylinositol-3 kinase (PI3K). Despite these similarities, signaling through IRS1 and IRS2 leads to distinct tumor cell outcomes in vitro and in vivo. In vitro, IRS1 regulates cell proliferation and growth and IRS2 regulates metabolism, survival and invasion. In vivo, Irs2 is a positive regulator of tumor metastasis, whereas Irs1 does not promote metastasis. The major objective of this thesis work was to further the understanding of the mechanism by which IRS2 signaling regulates tumor progression.
To investigate how IRS-1 and IRS-2 regulate distinct tumor cell outcomes, I examined the involvement of the microtubule cytoskeleton in IRS-dependent signaling. I determined that IRS2-mediated AKT activation is dependent upon an intact microtubule cytoskeleton, whereas IRS1-mediated AKT signaling occurs independently of microtubules. As a result, drugs that disrupt microtubules promote apoptosis in cells that signal through IRS2, but cells that signal through IRS1 are resistant to the effects of microtubule disruption. However, AKT inhibition sensitizes IRS1-dependent cells to apoptotic cell death upon microtubule disruption. From a clinical perspective, my studies identify IRS2 as a potential biomarker for the response of breast cancer patients to anti-microtubule drug therapy. To investigate further the mechanism of IRS2 contributions to tumor progression, I employed a mutagenesis approach to identify structural requirements of IRS2 for its function. I established that the ability of IRS2 to activate PI3K is necessary for its regulation of both invasion and tumor initiating cell (TIC) self-renewal. I also identified two independent regions within the IRS2 C-terminus that are required for invasion and self-renewal, respectively. Characterization of the invasion-promoting region identified BMP2-induced protein kinase (BMP2K) as an interacting protein. Suppression of BMP2K expression in mammary tumor cells disrupts IRS2-mediated tumor cell invasion. Taken together, my work advances the understanding of how IRS2 contributes to breast cancer progression and provides a molecular understanding for the development of novel approaches for the treatment of breast cancer and other malignancies that rely upon IRS2.
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Cocolakis, Eftihia. "The effect of activin/TGF [beta] signaling in mammary epithelial and breast cancer cells /." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103038.
Full textAbstract:
Activin and TGFbeta, members of the TGFbeta superfamily, are pluripotent cytokines that are expressed in virtually every cell of the body. These factors play diverse roles in the body such as regulating early development of the embryo, differentiation, extracellular matrix formation, hematopoiesis, angiogenesis and immune functions. TGFbeta superfamily signaling is transduced by heteromeric serine/threonine kinase receptors at the cell surface and the intracellular mediator, the Smad complex. Following activation of the receptors, there is recruitment and phosphorylation the Smads. As a result the Smad proteins accumulate in the nucleus, bind co-activators or repressors and elicit or suppress transcription of target genes.To date, the molecular signaling mechanisms for activin/TGFbeta in mammary gland growth and differentiation have not been fully elucidated. Our data identify a novel regulatory crosstalk mechanism by which activin/TGFbeta induced Smad signaling acts to antagonize Stat5 transactivation in mammary epithelial cells. We demonstrate an inhibitory effect of activin/TGFbeta on milk protein expression, specifically betacasein. We further show that activin/TGFbeta inhibitory effect upon betacasein expression is not due to changes in either Stat5 phosphorylation, translocation to the nucleus or binding on the Stat5 response element. We finally demonstrate that the Smads are required to block Stat5 transactivation by activin/TGFbeta and show that they are important mediators in activin/TGFbeta inhibitory response upon Stat5 target gene expression, in particular betacasein and cyclin D1. Finally, we unveil the mechanism by which these two signaling cascades antagonize their effects and find that activated Smads inhibit Stat5 association with its co-activator CBP, thus blocking Stat5 transactivation of its target genes. Thus, we define a novel crosstalk mechanism between two divergent signaling pathways that are involved in regulating mammary gland growth and differentiation.
Whereas the role of TGFbeta signaling in breast cancer has been well characterized, we sought out to study the role and mechanism of action of activin in the human breast cancer T47D cells. We found that activin treatment of T47D cells leads to a potent inhibition of cell growth. We further show that activin induces the Smad, the p38-mitogen activated kinase pathways and the p38 downstream target ATF2. Finally, using specific inhibitors to block p38 MAPK, activin-mediated cell growth inhibition is completely abolished. Together, these results define a novel signaling mechanism induced by activin in breast cancer cells.
Finally in an attempt to identify genes regulated by activin in breast cancer cells, we discover the death adaptor molecule RAIDD as a novel target of activin signaling. We show that RAIDD mRNA and protein levels are potently upregulated by activin. Using antisense-oligos directed against RAIDD, we show that RAIDD expression is necessary in mediating activin inhibition in breast cancer cells. Hence, we define the involvement of a new player in activin mediated cell growth inhibition.
Collectively, these studies reveal novel mechanisms of the activin/TGFbeta signaling cascade in normal mammary epithelial cells and breast cancer cells.
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Till, Kathleen June. "The biology of hairy-cell leukaemia, with particular reference to #alpha#-interferon." Thesis, University of Liverpool, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386848.
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Spence, Andrew J. "The Effect of Lactic Acid on Mast Cell Function." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3629.
Full textAbstract:
This study shows for the first time the effect that L-(+)-lactic acid has on mast cell activation. Lactic acid is a byproduct of anaerobic glycolysis and is associated with inflammatory environments such as wounds, tumors and, asthma. In this study, pre-treatment with lactic acid altered cytokine production by bone marrow-derived mast cells (BMMC). Specifically, lactic acid enhanced cytokine secretion following IgE cross-linking, but decreased IL-33 mediated cytokine production. These effects were altered by genetic background, since C57BL/6 mast cells demonstrated the aforementioned result, but lactic acid had no effect on IgE-mediated cytokine production in 129/SvJ mast cells. The affected cytokines included IL-6, TNF, MCP-1, MIP-1α, IL-13, and VEGF. Lactic acid pretreatment promoted a G0/G1 cell cycle arrest. Investigation into the IL-33 signaling pathway showed lactic acid decreased TAK1 and JNK phosphorylation, while increasing phosphorylated AKT levels. Blocking JNK and TAK1 with a small molecule inhibitor mimicked the effects of lactic acid. Interestingly, lactic acid significantly increased IL-33 mediated VEGF. An in vitro angiogenesis assay confirmed that mast cells were pro-angiogenic in a lactic acid-rich environment. Taken together, these data show that lactic acid impacts mast cell function, possibly promoting a pro-angiogenic, anti-inflammatory phenotype.
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Weng, Yaochung. "Measuring the effects of drugs on single cancer cell growth." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/72638.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student submitted PDF version of thesis.
Includes bibliographical references.
Understanding the effectiveness of a drug therapy on halting disease progression is an essential aspect of cancer biology. Conventional assays that study cell behavior after a drug intervention report the average response of a cell population which can mask the heterogeneity and dynamics of seemingly identical cells. Recently, many single-cell techniques have been developed, but there are currently no methods that can fully characterize the long-term effects of drug treatment on cancer cell growth. To accomplish such, we developed an instrument to measure single-cell growth before and after drug treatment. In order to achieve femtogram-level mass resolution, we employed the suspended microchannel resonator (SMR), a vacuum-packaged cantilever with an embedded channel. Here, we describe three implementations that involve different technologies (optical trap, mechanical trap, and dynamic ow trapping) to capture a cell for repeated measurements and to perform drug delivery. Applying the technique we developed based on the dynamic ow trapping, we were able to monitor one or more generations of a cancer cell before and after drug treatment. We investigated the growth of mouse leukemia cells in response to drugs that inhibit the mammalian target of rapamycin (mTOR) pathway, induce apoptosis, or prevent translational activity directly at the ribosome. Our method was able to discern a particular growth signature for each drug investigated and to discover a new phenotype in cells following mTOR inhibition. Furthermore, our data demonstrates that the instantaneous growth rate changes following a drug treatment could potentially predict the long-term inhibitory effect on cellular biogenesis and mass accumulation.
by Yaochung Weng.
Ph.D.