STAT3 is a critical oncogenic transcription factor, but how it becomes aberrantly activated in cancer is unclear. We have discovered a new pathway whose loss is associated with persistent STAT3 activation in human cancer. We found that the tumor suppressor miR-146b is a direct STAT3 target gene in normal breast epithelial cells. However, STAT regulation of miR-146b is subverted in tumor cells and is suppressed by promoter methylation, which is increased in primary breast cancers. Moreover, we show that miR-146b inhibits NF-κB-dependent IL-6 production, IL-6-dependent STAT3 activation, and IL-6/STAT3-driven functional phenotypes, thereby establishing a negative feedback loop. In addition, miR-146b expression appears to be deregulated in tumors with the highest levels of activated STAT3, and is positively correlated with patient survival. Our results indicate a new mechanism of crosstalk between STAT3 and NF-κB relevant to constitutive STAT3 activation in malignancy and the role of inflammation in oncogenesis.

This thesis is about new and improved computational methods to analyze complex biological data produced by advanced biotechnologies. Such data is not only very large but it also is characterized by very high numbers of features. Addressing these needs, we developed a set of methods and tools that are suitable to analyze large sets of data, including next generation sequencing data, and built transparent models that may be interpreted by researchers not necessarily expert in computing. We focused on brain related diseases. The first aim of the thesis was to employ the meta-server approach to finding peaks in ChIP-seq data. Taking existing peak finders we created an algorithm that produces consensus results better than any single peak finder. The second aim was to use supervised machine learning to identify features that are significant in predictive diagnosis of Alzheimer disease in patients with mild cognitive impairment. This experience led to a development of a better feature selection method for rough sets, a machine learning method.  The third aim was to deepen the understanding of the role that STAT3 transcription factor plays in gliomas. Interestingly, we found that STAT3 in addition to being an activator is also a repressor in certain glioma rat and human models. This was achieved by analyzing STAT3 binding sites in combination with epigenetic marks. STAT3 regulation was determined using expression data of untreated cells and cells after JAK2/STAT3 inhibition. The four papers constituting the thesis are preceded by an exposition of the biological, biotechnological and computational background that provides foundations for the papers. The overall results of this thesis are witness of the mutually beneficial relationship played by Bioinformatics in modern Life Sciences and Computer Science.

This dissertation elucidates several independent molecular mechanisms that function in astrocytes and glial tumor cells, and suggest that developmental and inflammatory signals may contribute to the development of brain tumors. First, we analyzed the mechanism of TIMP-1 activation in astrocytes and glioblastoma cells. TIMP-1 expression is activated by IL-1, which is the major neuroinflammatory cytokine, via simultaneous activation of IKK/NF-kB and MEK3/6/p38/ATF-2 pathways in primary human astrocytes. In contrast to astrocytes, TIMP-1 is expressed at lower levels in glioblastomas, and is not regulated by IL-1 due to either dysfunctional IKK/NF-kB or MEK3/6/p38/ATF-2 activation. Thus, we propose a novel mechanism of TIMP-1 regulation, which ensures an increased supply of the inhibitor after tissue injury to limit the ECM degradation. This mechanism does not operate in gliomas, and may in part explain the increased invasiveness of glioma cells.Inflammation has been associated with the development of several cancers, including glioblastoma multiforme. However, it has not been linked to other brain tumors. Here we show for the first time that inflammation is associated with oligodendroglioma tumors as pro-inflammatory cytokines, such as OSM, IL-6, MCP1, MIP1α, and MIP1β and inflammatory markers, such as ACT and COX-2, were expressed at higher levels in oligodendroglioma samples. In addition, cytokine-induced STAT3 signaling, but not NF-kB, is highly activated in the oligodendroglioma patients. Moreover, OSM promotes oligodendroglioma cell proliferation in vitro, and this effect is mediated through STAT3. In summary, oligodendroglioma tumors secrete and respond to inflammatory mediators, with OSM being the major cytokine that activates STAT3 to promote the growth of tumor cells, and express ACT and COX-2 as a hallmark of ongoing inflammation. Since STAT3 promotes the growth of oligodendroglioma, as well as glioblastoma cells, and also regulates gliogenesis, we studied molecular mechanisms of this process in an in vitro differentiation model. We turn our attention to the NFI family of transcription factors since they have recently emerged as novel regulators of the development of vertebral neocortex. We developed a stem cell-neural progenitor-astrocyte differentiation model, in which the generated astrocytes were characterized by proper morphology, increased glutamate uptake, and expression of early and late astrocyte markers. Moreover, we found that NFI-X and NFI-C but not NFI-A or NFI-B, control the expression of GFAP and SPARCL1, the markers of terminal differentiation of astrocytes.In summary, the three mechanisms of gene regulation we studied, provided new insights into astrocyte biology, with the important implications for understanding the basis leading to the development and progression of brain tumors.

YKL-40 is a secreted protein that is highly up-regulated in malignant glioblastoma (GBM). Its expression is correlated with the invasive nature of GBMs and poor diagnosis of patients (Nigro et al., 2005). Previous research has shown that in astrocytes and GBM cells, YKL-40 expression is regulated by two transcription factors, NFI-X3 and STAT3, which form a complex with each other (Singh et al., 2011). Here, we show that the N-terminal domain of NFI-X3 is sufficient and required for its interaction with STAT3. We also show that the DNA-binding domain of NFI-X3 is required to induce YKL-40 expression. Thus, the interaction of NFI-X3 with STAT3 may play a role in stabilizing the otherwise weak binding of NFI-X3 to the YKL-40 promoter. Collectively, the observations made in this study shed light on the mechanisms by which NFI-X3, in concert with STAT3 regulate YKL-40 expression.

In recent years, a number of nuclear transcription factors have been shown to be present in the mitochondria where they have distinct roles in regulating mitochondrial function. Signal Transducer and Activator of Transcription 3 (STAT3), classically activated by the JAK family of receptor associated tyrosine kinases to drive nuclear gene expression, is one such transcription factor with a unique mitochondrial role. There, it has been shown to support oxidative phosphorylation, regulate mitochondrial-encoded transcripts, and be key for the transformation and growth of a number of different cancers. Despite its well-characterized functional importance at the level of the mitochondria, the mechanism through which mitochondrial STAT3 acts and how it is regulated has not been as well studied. Using various cell culture models, we now show that mitochondrial STAT3 is dynamically regulated by oxidative stress and cytokine treatment in the acute setting. Under these conditions we have observed a rapid loss of mitochondrial STAT3 that recovers to baseline conditions with time. During this recovery phase we have noted that mitochondrial STAT3 becomes competent to bind to Cyclophilin D (CypD), the key regulator and activator of the mitochondrial permeability transition pore (MPTP). This is particularly the case with oxidative insults, which we believe may represent an important homeostatic mechanism for the cell. Intriguingly, chronic stimulation with certain stressors seems to increase mitochondrial STAT3 levels suggesting differential regulation in the acute versus chronic setting. The regulation of mitochondrial STAT3 levels by various stimuli points to a novel signaling pathway potentially linking mitochondrial responses with those of the cell. Unification of responses throughout the cell would seem to serve a clear adaptive advantage, particularly in coupling nuclear regulation with metabolic demands as dictated by the mitochondria. Extramitochondrial signaling, also known as the mitochondrial retrograde response, has emerged as an important homeostatic mechanism in lower organisms, but its signaling components have not been well characterized at the mammalian level. Our results point to a role for mitochondrial STAT3 in sensing cellular inputs, whereby its regulation and subsequent association with CypD may have implications in overall mitochondrial quality control. Though the inner workings of this signaling cascade are just beginning to be elucidated, they suggest the existence of a previously unappreciated pathway at the mitochondrial level.

No description available.

O câncer é um problema de saúde pública mundial, apresentando acréscimo na sua incidência a cada ano. O seu processo de evolução ainda não foi completamente desvendado, dificultando a elaboração de terapias adequadas. Na busca por um melhor prognóstico, pesquisas recentes têm discutido o papel das citocinas inflamatórias, do nicho perivascular e das células-tronco nos mecanismos de desenvolvimento e manutenção dos tumores malignos. Os tumores de glândula salivar representam uma pequena porcentagem das patologias malignas da região de cabeça e pescoço, podendo ocorrer em adultos e em crianças. O diagnóstico dificilmente é precoce e a taxa de sobrevida é extremamente baixa comparada aos demais tumores da região. Assim, este estudo teve como objetivo estudar as células provenientes dos tumores de glândula salivar do tipo adenoide cístico (CAC) e adenocarcinoma NOS (AdNOS) quanto ao seu perfil imunofenotípico, quanto à existência ou não de células-tronco tumorais nessa população, bem como investigar possíveis modificações na ativação de STAT3, Akt e ERK (moléculas envolvidas em vias de sinalização de manutenção do tumor), quando em contato com fatores secretados por células endoteliais. Foram coletados 5 CACs e 4 AdNOS, no Hospital da Universidade de Michigan (Ann Arbor, MI, EUA), durante 2010 e 2012. As células foram isoladas e caracterizadas em citometria de fluxo em P0 e P7, demonstrando um perfil de células CD44+ALDH+Lin- variando de 0,33 a 3,19% e 0,36 a 2,00%, respectivamente, entre 5 linhagens avaliadas. Na avaliação por western blotting, a e-caderina, o Snail e a actina de músculo liso foram ausentes em todos os tipos tumorais, enquanto que a citoqueratina 20 (Ck20) foi presente apenas nos AdNOS. Comparando os tumores com suas metástases, a presença de Ck20, p63 e β-catenina foi semelhante, enquanto que citoqueratina 7, a vimentina e o Bmi-1 foram maiores nas metástases. Tanto os AdNOS quanto CACs apresentaram receptores para IL-6, IL-8 e EGF. Foi observado que mediadores solúveis liberados pelas células endoteliais foram capazes de fosforilar STAT3, Akt e ERK em todas as células salivares estudadas, no entanto, a proteína recombinante humana IL-6, isoladamente, não foi capaz de ativar Akt. Orosferas foram geradas em todos os tipos tumorais, demonstrando o potencial de auto-renovação celular. Um maior número de esferas foi observado nas células metastáticas em relação às primárias. Células CD44+ALDH+, comparadas com CD44-ALDH-, geraram mais esferas, quando plaqueadas em alta densidade (5.000 células). No entanto, o inverso foi encontrado, quando uma única célula foi utilizada para o ensaio (p>0,05). Devido à dificuldade de obtenção e manipulação de células de tumores de glândula salivar, ainda há muito que se investigar mecanisticamente. Considerando a fosforilação de STAT3 na presença de IL-6, semelhante ao verificado em outros tumores, o uso de anticorpos contra IL-6, talvez sejam uma opção no futuro. / Cancer is a public health problem worldwide, with an increase in incidence every year. The process of its evolution is still not completely understood, hindering the development of appropriate therapies. In the search for a better prognosis, recent reports have discussed the role of inflammatory cytokines, perivascular niche and stem cells in the mechanisms of development and maintenance of malignant tumors. The salivary gland tumors represent a small percentage of malignancies of the head and neck and can occur in both adults and children. Early diagnosis is difficult and the survival rate is extremely low compared to other tumors in the same region. Thus, this study aimed to study cells from the adenoid cystic carcinoma (ACC) and adenocarcinoma NOS (AdNOS) tumors of salivary gland regarding its immunophenotypic profile and the existence or absence of tumor stem cells in this population, as well as investigate possible changes in the activation of STAT3, Akt and ERK (molecules involved in signaling pathways of tumor maintenance), when exposed to factors secreted by endothelial cells. ACCs (n=5) and AdNOS (n=4) were collected at the Hospital of the University of Michigan (Ann Arbor, MI, USA), during 2010 to 2012. Cells were isolated and characterized by flow cytometry at P0 and P7, showing a profile of ALDH+CD44+Lin- ranging from 0.33% to 3.19% and 0.36% and 2.00%, respectively, between 5 cell lines evaluated. In the protein profile, e-cadherin, Snail and SMA were absent in all tumor types. Ck20 was present only in AdNOS. Comparing primary tumors and their metastases, the presence of Ck20, and p63 β-catenin was similar, while Ck7, vimentin and Bmi-1 were higher in metastases. Both AdNOS as ACCs had receptors for IL-6, IL-8 and EGF. It was observed that soluble mediators released by endothelial cells were able to activate STAT3, Akt and ERK phosphorylation in all cells studied. However, recombinant human IL-6 alone was not able to activate Akt. Orospheres were generated in all tumor types, indicating the potential for cellular self-renewal. Highest number of spheres was observed in metastatic cells compared to primary. ALDH+CD44+ cells compared to ALDH-CD44- generated more spheres when plated in high density (5,000 cells), however, the opposite was found when one single cell seed was evaluated (p> 0.05). There is doubt if these cell markers would be consider for a stem cell model in salivary tumors. Due to the difficulty of obtaining and manipulating salivary gland tumor cells, there is still much to investigate mechanistically. As the phosphorylation of STAT3, in the presence of IL-6, was similar to that observed in other tumors, the use of antibodies against IL-6, may be an option in the future.

No description available.

Angiotensin II (Ang II) promotes vascular disease and hypertension in part by the formation of pro-inflammatory cytokines, oxidative stress and inflammation. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor known to play key roles in cytokine signaling and growth in immune cells. We tested the hypothesis that STAT3 plays an essential role in Ang II-induced vascular dysfunction and hypertension. Responses of carotid arteries from C57BL6 mice were examined in vitro after 22-hour incubation with vehicle or Ang II (10 nM) in the presence or absence of a small molecule inhibitor of STAT3 activation, S3I-201. The endothelium-dependent agonist acetylcholine (Ach) produced relaxation in arteries treated with vehicle and the response was inhibited by ~50% by Ang II (P<0.01). S3I-201 (10 πM) co-incubation prevented the Ang II-induced dysfunction. Relaxation to nitroprusside, an endothelium-independent agonist, was not altered in any group. Ang II increased vascular superoxide more than 2-fold (P<0.05) measured by chemiluminescence. S3I-201 (10 πM) prevented the Ang II induced increase of superoxide. Similar findings were obtained with STATTIC, a second small molecule inhibitor of STAT3 activation. In contrast to these findings, lipopolysaccharide (0.5 πg/ml)-induced endothelial dysfunction was not altered by S3I-201. Blood pressure and responses of carotid arteries and small resistance arteries within the brain were examined in C57BL6 mice with either saline or Ang II (1000 ng/kg/min) infused for 14 days via osmotic minipump, which were also treated with dimethyl sulfoxide (vehicle) or S3I-201 (5 mg/kg, IP, every two days). Infusion with Ang II increased systolic blood pressure compared to saline-infused animals (155±2 and 112±2 mmHg, respectively; P<0.001). S3I-201 reduced pressure slightly in saline infused mice but protected against Ang II-induced increase in pressure at 14 days (102±2 and 114±3 mmHg, respectively). Following systemic treatment with Ang II, carotid artery relaxation responses to Ach were significantly impaired compared to vehicle infused mice (72±3% and 101±1%, respectively, P<0.05). S3I-201 treatment significantly prevented Ang II-induced impairment (94±4%, P<0.05). Ang II treated mice exhibited 55% impaired dilator responses to Ach in small resistance arteries within the brain studied in vitro and S3I-201 treatment prevented most of this impairment (P<0.05). Vasorelaxation to nitroprusside was not altered in any group. In summary, these findings provide the first evidence that STAT3 plays an essential role in Ang II-induced vascular dysfunction and hypertension. Targeting STAT3 with small molecule inhibitors or other approaches may have beneficial effects during hypertension and other disease states in which Ang II contributes to vascular dysfunction (e.g. diabetes and aging).

The innate immune system is important in both acute and chronic infection. In this thesis, I investigated the effect of H. pylori infection on 1) DCs, key orchestrators of the immune system, and 2) autophagy, recently identified as an important component of innate immunity. I determined that H. pylori activates the STAT3 pathway in DCs, increasing DC maturation and inducing production of IL-10, IL-12p40 and TNF-α, without IL-12p70. This cytokine profile may favour an immunoregulatory response, promoting persistent H. pylori infection. In addition I determined that H. pylori’s VacA toxin induced autophagy, ROS production and Parkin aggregation which has been implicated in mediating autophagy in response to mitochondrial damage. Thus H. pylori alters these key effectors of innate immunity which may play a role in promoting its chronic infection and disease.