Décrypter les données omiques : importance du contrôle qualité. Application au cancer de l’ovaire Au cours des dix dernières années, la taille et la complexité des données biologiques ont littéralement explosé, et une attention particulière doit être portée au contrôle qualité. En effet, certaines données omiques (données génomiques et post-génomiques obtenues à haut débit) sont très incomplètes et/ou contiennent de nombreux biais et erreurs qu’il est facile de confondre avec de l’information biologiquement intéressante. Dans cette thèse, nous montrons que les interactions protéine-protéine issues de curation de la littérature et les interactions identifiées à haut débit sont beaucoup plus corrélées que ce qui est communément admis. Nous examinons l’interactome de la levure d’un point de vue original, en prenant en compte le degré d’étude des protéines par la communauté scientifique et nos résultats indiquent que cette corrélation s’estompe lorsqu’on se restreint aux protéines très étudiées. Ces observations nous permettent de proposer une méthode simple et fiable pour estimer la taille d’un interactome. Notre méthode conduit à une estimation d’au moins 37 600 interactions physiques directes chez S. cerevisiae, et montre que les évaluations précédentes sont trop faibles. Par ailleurs, nous étudions des données de séquençage nouvelle génération de l’ADN. Par une analyse des biais existant entre les short-reads alignés sur un brin ou sur l’autre du génome, nous mettons en évidence de nombreuses erreurs systématiques. De plus, nous observons de multiples positions présentant entre 20 et 40% de short-reads portant l’allèle variant : celles-ci ne peuvent pas être génotypées correctement. Nous proposons une méthode fiable pour appeler les génotypes à partir des données NGS qui permet de s’affranchir de ses difficultés. Enfin, nous appliquons cette méthode sur des données massives de séquençage d’exome de cellules saines et tumorales de 520 patientes atteintes du cancer de l’ovaire, produites par le consortium TCGA. Nous détectons en moyenne 30 632 variants germinaux par patiente. Parmi ces variants, nous identifions ceux les plus enclins à conférer un risque accru de développer la maladie : nous nous restreignons notamment aux variants induisant une perte de fonction de la protéine encodée et significativement plus présents chez les patientes que dans la population générale. Cela conduit à 44 SNVs par patiente en moyenne, répartis sur 334 gènes dans l’ensemble de la cohorte. Parmi ces 334 gènes, 42 ont été reportés comme impliqués dans la cancerogénèse, confirmant que la liste de candidats identifiés est fortement enrichie en gènes de susceptibilité au cancer de l’ovaire. En particulier, nos travaux confirment le rôle de suppresseur de tumeur de la protéine MAP3K8, très récemment proposée comme jouant un rôle clé dans d’autres cancers. / Deciphering omics data : on the importance of quality control. Application to ovarian cancer. Over the past 10 years, the size and complexity of biological data have exploded, and quality control is critical to interpret them correctly. Indeed, omics data (high- hroughput genomic and post-genomic data) are often incomplete and contain bias and errors that can easily be misinterpreted as biologically interesting findings. In this work, we show that literature-curated and high-throughput protein-protein interaction data, usually considered independent, are in fact significantly correlated. We examine the yeast interactome from a new perspective by taking into account how thoroughly proteins have been studied, and our results show that this bias can be corrected for by focusing on well- studied proteins. We thus propose a simple and reliable method to estimate the size of an interactome, combining literature-curated data involving well-studied proteins with high- hroughput data. It yields an estimate of at least 37,600 direct physical protein-protein interactions in S.cerevisiae, a significant increase over previous estimates. We then focus on next-generation DNA sequencing data. An analysis of the bias existing between short- eads aligned on each strand of the genome allows us to highlight numerous systematic errors. Furthermore, we observe many positions that exhibit between 20 and 40% of reads carrying the variant allele : these cannot be genotyped correctly.We then propose a method to overcome these biases and reliably call genotypes from NGS data. Finally, we apply our method to exome-seq data produced by the TCGA for tumor and matched normal samples from 520 ovarian cancer patients. We detect on average 30,632 germline variants per patient. Though an integrative approach, we then identify those which are likely to increase cancer risk : in particular, we focused on variants inducing a loss of function of the encoded protein, and selected those that are significantly more present in the patients than in the general population. We find 44 SNVs per patient on average, impacting 334 genes overall in the cohort. Among these genes, 42 have been previously reported as involved in carcinogenesis, confirming that our list is highly enriched in ovarian cancer susceptibility genes. In particular, our results confirm the tumor suppressor role of the MAP3K8 protein, recently identified in other types of cancer.

Made available in DSpace on 2014-06-11T19:29:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-29Bitstream added on 2014-06-13T20:39:41Z : No. of bitstreams: 1 buitragosanchez_f_me_botfm.pdf: 280716 bytes, checksum: a974f556f8eb7fd716a4968b04ed954a (MD5) / Fundação de Ensino e Pesquisa em Ciências da Saúde (FEPECS) / A super-expressão do HER-2 tem sido associada a maior agressividade biológica do tumor e a resistência a alguns tipos de tratamento. O objetivo deste trabalho é avaliar o valor prognóstico da super-expressão do HER-2, nas pacientes portadoras de câncer de mama. Estudo coorte que analisou 90 pacientes com diagnóstico de carcinoma ductal infi ltrante de mama atendidas no Hospital Regional de Sobradinho no período de 1 de Janeiro de 2005 até o 31 de dezembro de 2010. A investigação da super-expressão do HER-2 foi realizada por técnica de Imunohistoquimica. As pacientes foram divididas em dois grupos: Grupo A presença de super-expressão do HER-2 (HER-2 positivo) e grupo B ausência de super-expressão (HER-2 negativo). Ambos os grupos foram comparados às características clinicas, patológicas, eventos adversos, sobrevida global e sobrevida livre de doença. A prevalência da presença do HER-2 na amostra foi de 15,55%. A meia de idade no momento do diagnóstico, o tamanho do tumor, o estado menstrual, o estádio clinico e estado linfonodal não foram fatores signifi cativos de prevalência para a presença do HER-2 positivo. O receptor de estrogênio (p=0,0356), receptor de progesterona (p=0,0059) e mostraram associação signifi cativa com o HER-2. A análise multivariada mostrou que a prevalência do HER-2, em pacientes com câncer é 2,92 vezes maior naqueles com receptor de estrogênio negativo (p= 0,0007) e 8,84 vezes maior naqueles com receptor de progesterona positivo (p< 0,0001). A sobrevida livre de doença e sobrevida global é igual em pacientes com HER-2 positivo. A sobrevida livre de doença para pacientes com axila com mais de 4 linfonodos comprometidos é menor (p=0,0009) assim como a sobrevida global (p<0,0001). O tamanho tumoral também é fator adverso mostrando que tumores maiores de... / The overexpression of HER-2 has been associated with increased aggressiveness of the tumor and resistance to some types of treatment. The objective of this study is to evaluate the prognostic value of overexpression of HER-2 in patients with breast cancer. A cohort study that analyzed 90 patients diagnosed with infi ltrating ductal carcinoma breast treated at the Regional Hospital Sobradinho the period 1 January 2005 until December 31, 2010. The investigation of the presence of overexpression of HER-2 was performed by immunohistochemical techniques. The patients were divided into two groups: Group A who had the overexpression of HER-2 and the B group who had no overexpression and compared the clinical, pathological, adverse events, overall survival and disease-free survival. The prevalence of the presence of HER-2 in the sample was 15.55%. The age at diagnosis, tumor size, menopausal status, clinical stage and lymph node status were not signifi cant factors prevalence for the presence of HER-2. The estrogen receptor (p = 0.0356), progesterone receptor (p = 0.0059) and showed signifi cant association with HER-2. Multivariate analysis showed that the prevalence of HER-2 in cancer patients is 2.92 times higher in those with estrogen receptor negative (p = 0.0007) and 8.84 times higher in those with progesterone receptor positive (p < 0.0001). The disease-free survival and overall survival is equal in patients with HER-2 positive. The disease-free survival for patients with more than 4 axillary lymph nodes is lower (p = 0.0009) and overall survival (p <0.0001). The tumor size is also an adverse factor showing that tumors larger than 2 cm have a lower disease-free survival (p = 0.0103) and shorter overall survival (p = 0.0103). The presence of over-expression of... (Complete abstract click electronic access below)

<div>Prostate cancer (PCa) is the second leading cause of cancer related deaths in American men. In this study, I identify two combinational therapeutics to treat PCa – the combination of enzalutamide and simvastatin, and the combination of GSK126 and metformin, both of which strongly suppress PCa cell growth in vitro and in vivo via inhibiting androgen receptor (AR), an important oncogenic driver for the PCa progression. Simvastatin leads to more AR degradation when combined with enzalutamide. For the combination of GSK126 and metformin, the interaction between enhance of zeste homolog2 (EZH2) and AR is interrupted by GSK126, re-sensitizing EZH2 to metformin. Meanwhile, GSK126 inhibits EZH2’s activity. </div><div><br></div><div>Polo-like kinase 1 (PLK1), a cell cycle regulator, is usually overexpressed in non-small-cell lung cancer (NSCLC). Here, we report that PLK1 overexpression promotes the development of Kras^G12D and Trp53^fl/fl (KP)-driven lung adenocarcinoma (LADC). KP mice harboring transgenic PLK1 (KPPI) display heavier tumor burden, poorer tumor differentiation, and lower survival than KP mice. Mechanistically, PLK1 overexpression enhances the activity of MAPK pathway, via upregulating RET expression in a kinase-dependent manner. Supporting our findings, PLK1 knockout in KP mice reduces RET gene expression, inhibits MAPK pathway activity, and strongly delays LADC development. Therefore, these data reveal that PLK1 functions as an oncogene in KP-driven LADC.</div><div> </div><div><br></div>

On average, 65 Canadian men will be diagnosed with prostate cancer (PC) every day, making it the most common male cancer in Canada. Despite the prevalence, the etiology of PC is unknown. Evidence nonetheless supports the role of prostate cancer stem cells (PCSCs) in PC initiation and metastasis. In spite of almost a decade worth of research on PCSCs our knowledge on their biology remains fragmented. By taking advantage of the availability of DU145 cell-derived PCSCs in our laboratory, this thesis research focuses on investigating the unique properties of PCSCs and their function in promoting PC tumorigenesis. We identified two PCSC-specific proteins, ALDH3A1 and CNTN1. In mouse models of xenograft tumors, ALDH3A1 was expressed at higher levels in PCSC-derived tumors than in DU145 non-PCSC-produced tumors and in lung metastases than local tumors. In clinical settings, elevation of ALDH3A1expression was observed from normal prostate tissues to carcinomas and from local PCs to the paired lymph node metastases. Additionally, ALDH3A1 was clearly detected in bone metastases. Similar to ALDH3A1, CNTN1 expression associates with PC progression and biochemical recurrence following radical prostatectomy. The clear presence of CNTN1 in lymph node and bone metastases was also demonstrated. Furthermore, CNTN1 expression promoted PC metastasis to the lungs and tumor initiation in NOD/SCID mice. Mechanistically, CNTN1 increased AKT activation and reduced E-cadherin expression. Collectively, our research revealed important roles of both PCSC proteins in promoting PC tumorigenesis and progression. PC develops chemotherapy resistance in which PCSCs play a major role. In supporting this knowledge, we demonstrated that PCSCs are innately more resistant to the chemotherapeutic drugs, etoposide and docetaxel and that this resistance was in part attributable to their enhanced DNA damage response. Taken together, the findings of this thesis advances our knowledge on two specific PCSC markers and their association with prostate cancer progression and metastasis. As well as to the mechanism whereby PCSCs promote resistance to chemotherapeutic drugs. / Thesis / Doctor of Philosophy (PhD)

Title: Investigation of RRM2 as a potential therapeutic target against glioblastoma Supervisors: Dr. Sven Nelander and Mr. Sathishkumar Baskaran. Department: Department of Immunology, genetics and pathology (IGP), Uppsala University   Glioblastoma (GBM) is the most malignant form of glioma and associated with high proliferation rate, necrosis and highly invasive nature. Current treatment includes tumor resection followed by combination of radiotherapy and chemotherapy with temozolomide (TMZ). Despite of combination therapy, GBM exhibits dismal prognosis and mean survival rate of patients is only 3.3 % at 2 years and 1.2 % at 3 years. Therefore, there is an increasing demand of identifying new therapeutic targets against GBM. In this project, we studied the function of RRM2 gene as a potential therapeutic target in two patients derived GBM cell lines (GC). By knocking down RRM2 using short interfering RNAs, the viability of cells and proliferation was significantly reduced in both the GC. The cause of cell death was due to induction of apoptosis by the treatment in GC. Treatment of COH29, an inhibitor of RNR, induced cell death at therapeutically relevant dose in GC. Our results indicate that RRM2 has a significant role in GBM cell growth/proliferation. More evaluation must be performed in both in-vitro and in-vivo to pursue RRM2 as a molecular therapeutic target against GBM.

Thesis (M.S.)--Boston University, 1956 / This thesis represents one part of a group study conducted jointly by four Boston University social work students. The purpose of this group study is to examine first same of the social and emotional factors that affect the rehabilitation of cancer patients, and second, the role of social service in this rehabilitation. The study is based on the cases comprising the Harvard Medical School project.

Although there are hereditary risk factors strongly associated with breast cancer, only a small percentage of breast tumors can be attributed to these. Instead, it is believed that 85-90% of breast cancers are primarily of environmental origin. Polycylic aryl hydrocarbons (PAHs) are environmental carcinogens derived from combustion including fossil fuels. PAHs bind to a cellular aryl hydrocarbon receptor (AhR) that mediates downstream events leading to cellular transformation. Previous work in our laboratory used the prototypical PAH 7,12-dimethylbenz[a]anthracene (DMBA) to form tumors in mouse mammary glands that had constitutive AhR activation, increased Wnt signaling, and strong induction of the CK2 subunit CK2α (Currier, Solomon et al. 2005). Wnt, an important developmental pathway, is implicated in several cancers (Dominguez, Sonenshein et al. 2009). CK2 is a highly promiscuous, constitutively active serine/threonine kinase that is over-expressed in every cancer that has been examined for its presence (Meggio and Pinna 2003). Data from the DMBA-induced mouse tumors demonstrated that these factors may be involved in carcinogen-induced breast cancer, but their role in tumor development is uncertain. The hypothesis underlying this project was that CK2 and the Wnt pathway would be involved in early changes in mouse mammary and liver tissues in animals exposed to DMBA. We used qPCR, Western blotting, and immunohistochemistry to look at changes in Wnt pathway components and known Wnt-dependent genes, and CK2 subunits in mammary and liver tissues one and two weeks after DMBA exposure. Liver tissue was analyzed along with mammary gland tissue because the liver is the site of DMBA metabolism. Our results showed no change in liver or mammary gland morphology at these time points. There was induction of the AhR gene targets cyp1a1 and cyp1b1 in liver tissue but not in mammary gland. Liver also had evidence of Wnt pathway activation. Mammary glands did not have a strong AhR response but did show Wnt induction at two weeks post-exposure, suggesting that DMBA was affecting the liver before the mammary glands. CK2α had an unexpected early decrease in protein expression at one week in liver, which at two weeks resolved to the same levels as control tissue. In mammary glands, CK2α expression levels were the same as control at one week and decreased at two weeks, again suggesting a slower response than liver. Interestingly, CK2β was markedly overexpressed in mammary glands at the two week time-point. These results suggest there is a role for both Wnt and CK2 in early DMBA-generated tissue changes. It is still unclear if these pathways are separately affected by DMBA or if one initiates the other. Further experimentation, possibly in cell culture using inhibitors and siRNA, are called for to better understand these findings.

Angiogenesis expands the vascular network during normal development and in response to angiogenic stress. Dysregulation of this dynamic process contributes to the pathogenesis of many diseases including retinopathies. The α2β1 integrin, a collagen and laminin receptor which is linked to risk of vascular retinopathy, plays an important yet incompletely understood role in angiogenesis. In this dissertation, I employ multidisciplinary approaches to examine the function of this integrin during both pathological and developmental angiogenesis in the retina. The major goal is to contribute clinically relevant knowledge through mechanistic investigation of the link between α2β1 integrin in vascular retinopathies and careful exploration of this integrinâs role in angiogenesis. The central questions addressed in this thesis are, 1) does the α2β1 integrin contribute to the progression of retinopathies, and through what mechanism? And, 2) how does α2β1 integrin interface with the major angiogenesis pathways, and does that explain the divergent effects of α2-integrin deletion in different vascular beds? Using the oxygen-induced retinopathy (OIR) model for retinopathy of prematurity (ROP) on wild type and α2-null mice, I elucidated the role of α2β1 integrin in both endothelial cells as well as in the retinal microenvironment. I uncover a novel, potentially mechanistically important role for the integrin in regulating retinal Müller cell function. To clarify the role of α2β1 integrin in angiogenesis, I use in vitro, in vivo and in silico methods to characterize wild type and α2-null mice during postnatal development of the retinal vasculature. I develop a hybrid mathematical model to simulate cell signaling and vascular morphology in the developing postnatal murine retina. Using this model, I study how the VEGF-notch signaling system directs the development of morphological features including, retinal vascularization, plexus density, and plexus irregularity. I also use the model to predict how crosstalk to the VEGF-Notch axis from other angiogenic signals affects vascular phenotypes. Finally, I use the computational model as a platform for evaluating proposed signaling relationships between α2β1 integrin and the VEGF-Notch axis and present a molecular model which may explain how α2-integrin deletion causes disparate vascular phenotypes in different vascular microenvironments.

Dysregulation of receptor tyrosine kinases (RTKs) contributes to cellular transformation and cancer progression by disrupting key metabolic signaling pathways. The EPHA2 RTK is overexpressed in aggressive forms of breast cancer, including the HER2+ subtype, and correlates with poor prognosis. However, the role of EPHA2 in tumor metabolism remains unexplored. This thesis sought to determine the mechanisms by which EPHA2 ligand-independent signaling promotes tumorigenesis in the absence of its prototypic ligand, ephrin-A1. We integrated in vivo and in vitro models of HER2-overexpressing breast cancer to demonstrate that loss of ephrin-A1 leads to upregulated glutamine metabolism and lipid accumulation that enhanced tumor growth. Global metabolic profiling of ephrin-A1-null, HER2-overexpressing mammary tumors revealed a significant increase in glutaminolysis, a critical metabolic pathway that generates intermediates for lipogenesis. Pharmacologic inhibition of glutaminase activity reduced tumor growth in both ephrin-A1-depleted and EPHA2-overexpressing tumor allografts in vivo. Mechanistically, we show that the enhanced proliferation and glutaminolysis in the absence of ephrin-A1 was attributed to increased RhoA-dependent glutaminase activity. EPHA2 depletion or pharmacologic inhibition of Rho, glutaminase, or fatty acid synthase abrogated the proliferative effects of ephrin-A1 knockdown. Together, these findings highlight a novel, unsuspected connection between the Ephrin-A1/EPHA2 signaling axis and tumor metabolism, and suggest potential new therapeutic targets in cancer subtypes exhibiting glutamine dependency.

Benign prostatic hyperplasia (BPH) is a common, progressive chronic disease. Inflammation is associated with prostatic enlargement and resistance to 5?-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-?B) pathway is linked to both inflammation and ligand-independent prostate cancer progression. Most patients initially respond to 5ARI therapy; however, failure is common. To address why patients fail therapy we used transition zone tissue samples from patients with a wide range of American Urological Association symptom score (AUASS) from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. NF-?B activation and androgen receptor variant (AR-V) expression were quantified. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-?B activity, AR-FL, and AR-variant 7 (AR-V7). We determined that canonical NF-?B signaling was found to be upregulated in late versus early stage BPH. Elevated expression of AR-V7 was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS. Forced activation of canonical NF-?B in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-?B and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment. To understand why NF-?B and AR-V7 maintained viability within 5ARI treatment we examined the levels of 5?-reductase enzymes (SRD5A1, SRD5A2, SRD5A3). We determined that SRD5A2 is upregulated in more advanced BPH. SRD5A2 was significantly associated with AUASS and patients on a 5ARI. AR-FL and AR-V7 expression increased SRD5A2 expression whereas forced NF-?B activation increased all SRD5A isoforms. In summary, activation of NF-?B and AR-V7 in the prostate is associated with increased disease severity. Increased BPH severity in patients correlates with SRD5A2 expression. De novo synthesis of androgens and AR-V7 expression is inducible in human prostate cells by forced activation of NF-?B. NF-?B and AR-V7 upregulate SRD5A2 resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy.