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.

Cancer cells take more glucose to provide energy and phosphoryl intermediates for cancer progression. Meanwhile, energy-provider function of mitochondria in cancer cells is disrupted. This phenomenon is so-called Warburg effect, which is discovered over eighty years ago. The detail mechanisms for Warburg effect are not well defined. How glycolytic enzymes contribute to cancer progression is not well known. PKM2 is a glycolytic enzyme dominantly localized in the cytosol, catalyzing the production of ATP from PEP. In this study, we discovered that there were more nuclear PKM2 expressed in highly proliferative cancer cells. The nuclear PKM2 levels are correlated with cell proliferation rates. According to our microarry analyses, MEK5 gene was upregulated in PKM2 overexpression cells. Our studies showed that PKM2 regulated MEK5 gene transcription to promote cell proliferation. Moreover, nuclear PKM2 phosphorylated Stat3 at Y705 site using PEP as a phosphoryl group donor to regulate MEK5 gene transcription. Our study also showed that double phosphorylated p68 RNA helicase at Y593/595 interacted with PKM2 at its FBP binding site. Under the stimulation of growth factors, p68 interacted with PKM2 to promote the conversion from tetrameraic to dimeric form so as to regulate its protein kinase activity. Overexpression PKM2 in less aggressive cancer cells induced the formation of multinuclei by regulating Cdc14A gene transcription. Overall, this study presents a step forward in understanding the Warburg effect.

Myeloid-derived suppressor cells (MDSC) are a major component of the immune suppressive network that develops during cancer. MDSC down-regulate immune surveillance and antitumor immunity and facilitate tumor growth. The ability of MDSC to suppress T cell responses has been documented; however the mechanisms regulating this suppression remain to be understood. This work proposes a biological dichotomy of MDSC regulated by the tumor microenvironment. In peripheral lymphoid organs MDSC cause T-cell non-responsiveness that is antigen-specific. These MDSC have increased expression of NOX2, enabling them to produce large amounts of reactive oxygen species. Since the transcription factor STAT3 is substantially activated in MDSC, its potential role in upregulation of NOX2 expression was investigated. Over-expression of a constitutively active form of STAT3 increases expression of NOX2 subunits, whereas attenuation of STAT3 activity leads to decreased expression of NOX2. The significance of NOX2 in ROS generation is demonstrated in mice devoid of NOX2 function; NOX2- deficient MDSC are unable to inhibit antigen-induced activation of T cells. In contrast, MDSC within the tumor microenvironment have a diminished potential to generate ROS but acquire expression of arginase and inducible nitric oxide synthase, enzymes plicated in T cell non-responsiveness. Upregulation of these enzymes results in MDSC ability to inhibit lymphocyte response in absence of antigen presentation. The tumor microenvironment also promotes the differentiation of MDSC to tumor associated macrophages. Hypoxia is an exclusive feature to the tumor microenvironment and we investigated its involvement in the properties of MDSC at the tumor site. Exposure of spleen MDSC to hypoxia converts MDSC to non-specific suppressors and induces a preferential differentiation to macrophages. Stabilization of HIF-1!, a transcription factor activated by hypoxia, induces similar changes in MDCS as hypoxic exposure. Finally, ablation of HIF-1! prevents MDSC from acquiring factors that enable the suppression of T cells in absence of antigen. These findings help to expand our understanding of the biology of MDSC and suggest a regulatory pathway of myeloid cell function exclusive to the tumor microenvironment. They may also open new opportunities for therapeutic regulation as we now should take into consideration how systemic location affects the function of MDSC.

Few transcription factors (TFs) have been studied in the context of an integrative analysis incorporating genomic datasets from diverse genome regulatory mechanisms. Such an analysis allows the testing of specific regulatory associations in an unbiased and comprehensive manner. The promoter binding TF complex NF-Y regulates a diverse set of constitutive, inducible, developmental, oncogenic and tissue-specific genes. Using cancer models, ChIP-Seq, shRNA, and genomics, I have undertaken a genome-wide study of NF-Y. NF-Y binds to not only promoters but also extensively to enhancers, select classes of repetitive elements, inactive chromatin domains and insulators. NF-Y is a “pioneer”-like factor able to access its motif within closed, transcriptionally inactive chromatin domains. NF-Y pervasively associates with FOS, usually in the absence of JUN and the AP-1 motif, and with a group of growth controlling oncogenic TFs. I also show that NF-Y asymmetrically binds to its motif and stereo-aligns with specific TFs and their motifs. My results indicate that NF-Y is not merely a commonly-used, proximal promoter TF, but rather functions at a more diverse set of genomic elements. The dynamics of TF occupancy, cis-regulatory element (CRE) usage and their linkage to gene expression during a differentiation process, from a genome-wide perspective, is poorly understood and is critical to the understanding of fundamental aspects of development and disease. I utilize a model of inflammation-mediated oncogenic transformation, siRNA, ChIP-Seq, FAIRE-Seq, and microarrays to study the genomic aspects of transformation driven by Src-mediated activation of the inflammatory TF STAT3. I show that CRE usage is static, even in the presence of induced STAT3 activity, and large-scale transcriptional and phenotypic changes. STAT3 induced occupancy is tightly associated with FOS, pre-existing CREs, and does not create CREs de novo. I also highlight a putative role of TSC22D3 in inhibiting an epigenetic switch and in STAT3 and AP-1 factors driving the embryonic-like and bone-like phenotypes of breast cancer. The research presented here suggests that phenotypic alterations occurring during disease are not driven by large-scale perturbations of CRE usage. Overall, this dissertation provides an invaluable resource of genome-scale datasets within cancer models that will assist in future endeavors of scientific discovery.

Overactivity of platelet-derived growth factor receptor (PDGFR) is a frequent event in many types of solid tumors. Therefore, it is of great importance to uncover the mechanisms that regulate PDGF/PDGFR signalling, to develop efficient inhibitors targeting this pathway. The first step of downregulation of PDGFR activity upon ligand binding is internalization; thus we investigated how endocytosis pathways affect PDGFR signaling. We showed that in Ras-transformed fibroblasts, the internalization of PDGFR is shifted from the routine clathrin-dependent endocytosis to macropinocytosis, which results in enhanced PDGFR activity and subsequent downstream signalling, promoting anchorage-independent growth. We were also interested in how intracellular trafficking regulates signalling attenuation of PDGFR. We found that His-domain containing protein tyrosine phosphatase (HD-PTP) positively regulates phosphorylation level of the ubiquitin-ligases c-Cbl and Cbl-b; consistently, silencing of HD-PTP led to a decreased level of PDGFR ubiquitination (paper II). Consequently, internalized PDGFR could not be sorted properly and escaped degradation. This resulted in enhanced activation of phospholipase C γ (PLCγ) and changed kinetics of signal transducer and activator of transcription (STAT) 3 signalling, which further increased colony formation of HD-PTP silenced cells in soft agar, indicating a tumor suppressor role of HD-PTP. Activation of PDGFR leads to stimulation of downstream pathways. We identified Fer kinase as a critical signal transducer downstream of PDGFR in a proteomic screen. We showed that Fer kinase is essential for PDGF-induced STAT3 activation; as a result (paper III), Fer depletion severely blunted the ability of PDGFR signalling to promote anchorage-independent growth in soft agar and delayed tumor initiation in a mouse model. The crosstalk between host and tumor plays a critical role in tumor progression. At present most anti-cancer drugs are targeting tumor cells; we were interested in how targeting tumor host cells affects the efficacy of anti-tumor therapy. We found that selective PDGFRβ inhibition in host cells exerted tumor inhibitory effects on growth and vascularization of tumors with autocrine PDGF signaling, whereas tumors lacking such stimulation show only minor response on tumor growth (paper IV). Meanwhile, we demonstrated that PDGF/PDGFRβ signalling promotes expression of NG2, a marker for pericytes.

The signal transducer and activator of transcription-3 (Stat3) is a latent cytoplasmic protein that is activated through phosphorylation of tyrosine-705 by a number of receptor and non-receptor tyrosine kinases. This leads to Stat3 dimerization by reciprocal SH2-ptyr interactions, followed by translocation to the nucleus to initiate transcription of genes involved in cell growth, survival, and differentiation. Many of these signaling molecules known to activate Stat3 concentrate in specialized plasma membrane microdomains called caveolae, and are sequestered in an inactive state to the caveolin scaffolding domain (CSD) of the main caveolae resident protein, caveolin-1 (cav1). Since many of these signaling molecules are known, potent Stat3 stimulators, we set out to examine the effect of cav1 upon Stat3 activity. To this effect, cav1 was downregulated using a cholesterol chelator (methylcyclodextrin), or an antisense approach. Since we previously found that cell density can dramatically activate Stat3, all experiments were conducted at several densities. The results show that cav1 downregulation causes an increase in Stat3-tyr705 phosphorylation at all densities examined. We next examined the effect of cav1 upregulation upon Stat3 activity by transfecting an EGFP-cav1 construct. The results revealed that cav1 overexpression using this construct reduces Stat3 activity and induces apoptosis, which can be overcome by expression of a constitutively active form of Stat3. Finally, by expressing a Stat3 shRNA with an adenovirus vector, we demonstrated that Stat3 downregulation leads to an increase in cav1 levels. These results reveal the presence of a potent, negative regulatory relationship between cav1 and Stat3 phosphorylation. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2008-11-09 20:53:54.787

Advances in anti-cancer drug development have vastly improved cancer treatment strategies over the past few decades. Chemotherapeutic agents are now being replaced with targeted therapies that have much greater potency and far fewer unpleasant side effects. At the center of this, cell signaling pathways have been targeted as they moderate gene expression, control proliferation and are often dysregulated in cancer. The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes in response to extracellular signals. Of the seven mammalian members described to date, Stat3 has received particular attention, as it regulates the expression of genes involved in a variety of malignant processes including proliferation, survival, migration and drug resistance. Aberrant Stat3 activation has been observed in a number of human cancers, and its inhibition has shown promising anti-tumour activity in cancer cells with elevated Stat3 activity. Thus, Stat3 has emerged as a promising target for the development of cancer therapeutics. While Stat3 signaling can be inhibited by targeting upstream regulators of Stat3 activation (such as Janus kinase 2), direct inhibition of Stat3 protein may offer improved response, larger therapeutic windows for treatment and fewer side effects. The work presented within this thesis is focused on optimizing known Stat3 inhibitor S3I-201, a small molecule Stat3 SH2 domain binder that was discovered in 2007. We have performed an extensive structure activity relationship study that has produced some of the most potent Stat3 inhibitors in the scientific literature. These compounds showed high-affinity binding to Stat3’s SH2 domain, inhibited intracellular Stat3 phosphorylation and selectively induced apoptosis in a number of cancer cell lines. Lead agents further inhibited tumour growth in xenograft models of human malignancies and had favourable pharmacokinetic and toxicity profiles.

Advances in anti-cancer drug development have vastly improved cancer treatment strategies over the past few decades. Chemotherapeutic agents are now being replaced with targeted therapies that have much greater potency and far fewer unpleasant side effects. At the center of this, cell signaling pathways have been targeted as they moderate gene expression, control proliferation and are often dysregulated in cancer. The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes in response to extracellular signals. Of the seven mammalian members described to date, Stat3 has received particular attention, as it regulates the expression of genes involved in a variety of malignant processes including proliferation, survival, migration and drug resistance. Aberrant Stat3 activation has been observed in a number of human cancers, and its inhibition has shown promising anti-tumour activity in cancer cells with elevated Stat3 activity. Thus, Stat3 has emerged as a promising target for the development of cancer therapeutics. While Stat3 signaling can be inhibited by targeting upstream regulators of Stat3 activation (such as Janus kinase 2), direct inhibition of Stat3 protein may offer improved response, larger therapeutic windows for treatment and fewer side effects. The work presented within this thesis is focused on optimizing known Stat3 inhibitor S3I-201, a small molecule Stat3 SH2 domain binder that was discovered in 2007. We have performed an extensive structure activity relationship study that has produced some of the most potent Stat3 inhibitors in the scientific literature. These compounds showed high-affinity binding to Stat3’s SH2 domain, inhibited intracellular Stat3 phosphorylation and selectively induced apoptosis in a number of cancer cell lines. Lead agents further inhibited tumour growth in xenograft models of human malignancies and had favourable pharmacokinetic and toxicity profiles.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 5, 2008). Includes bibliographical references.

TEÓFILO, C. R. Investigação da associação entre os mirnas 10a, 17, 29c e 31 e a expressão de il-6 e stat3 em lesões orais potencialmente malignas, no câncer bucal e campo de cancerização. 2017. 105 f. Tese (Doutorado em Odontologia) - Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-05-02T12:28:15Z No. of bitstreams: 1 2017_tese_crteofilo.pdf: 2960055 bytes, checksum: 9b053dd115c86e0f4667ca5b49068d3c (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-05-02T12:28:22Z (GMT) No. of bitstreams: 1 2017_tese_crteofilo.pdf: 2960055 bytes, checksum: 9b053dd115c86e0f4667ca5b49068d3c (MD5) / Made available in DSpace on 2017-05-02T12:28:22Z (GMT). No. of bitstreams: 1 2017_tese_crteofilo.pdf: 2960055 bytes, checksum: 9b053dd115c86e0f4667ca5b49068d3c (MD5) Previous issue date: 2017-02-24 / Oral cavity malignant neoplasms are a serious public health problem in Brazil and in the world. The most common histologic subtype in the mouth are squamous cell carcinoma (SCC). Often, before the onset of SCC, there are clinical and histological changes with a greater potential for malignant transformation, called potentially malignant disorders (PMD). Multiple molecular factors may contribute to the development of this neoplasms. Signal Transducers and Activators of Transcription-3 (STAT3) is a cytokine-activated signal transducer, which may be directly active in neoplastic. High levels of IL-6, a possible STAT3 activator, have been associated with poor prognosis in SCC. MicroRNAs (miRNAs) are a group of RNAs that act as gene-regulator in the post-transcriptional phase through the degradation or silencing of messenger RNAs. The present study aimed to quantify microRNAs miR-10a, miR-17, miR-29c and miR-31 in the field effect area of oral dysplasia and SCC. Additionally, it is intended to evaluate the expression of IL-6 and STAT3 in these lesions. For this, samples of 47 SCC, 21 PMD and 21 controls were collected. In the patients with SCC or PMD, in addition to the lesion area, a normal mucosa sample was collected, 1 cm away from the lesion. miRNAs were analyzed by real-time PCR and STAT3 and IL-6 by immunohistochemistry. The SCC sample was, most of them, moderately differentiated (91.5%), with growth in nets (73%) and non-keratinizing (52.6%). Dysplasia presented, more frequently, mild (52%), and 42.8% of them showed intense subepithelial inflammation. The mean age of the patients in the three groups was 52 ± 21 years. Males were predominant in the SCC group (67.3%) and female in DPM (57.1%). The most frequent localization site for both groups was tongue. Hyperexpression of all studied miRNAs were observed in SCC and PMD, as well as in the perilesional regions. Evaluating SCC, all the miRNAs studied were correlated with each other, while in SCC perilesion increase of miR-31 was accompanied by elevation of miR-17 levels, as well as hyperexpression of miR-29c was accompanied by miR-10a increase. There was an increase of miR-17 and miR-29c in SCC compared to PMD. In PMD, the increase of miR-10a was accompanied by an increase of miR-17 and miR-29c; a similar situation occurred between miR-31 and miR-17. In PMD perilesion, miR-10a and miR-29c presented positive correlation. High expression of IL-6 was obtained in 100% of the SCC and PMD samples. STAT3 presented positivity for 84.6% of SCC samples and 88.2% of DPM samples. Positive correlation between miR-29c and miR-10a was found in SCC, in dysplasias and perilesional areas, indicating that hyperexpression of these microRNAs is an early event in the oral carcinogenesis. / As neoplasias malignas de cavidade oral constituem um sério problema de saúde pública no Brasil e no mundo, sendo o tumor maligno mais comum em boca o Carcinoma de Células Escamosas (CCE). Frequentemente, antes do surgimento do CCE, ocorrem alterações clínicas e histológicas com maior potencial de transformação maligna, chamadas desordens potencialmente malignas (DPM). Fatores moleculares podem contribuir para o desenvolvimento dessa neoplasia. O Signal Transducers and Activators of Transcription-3 (STAT3) é um transdutor de sinais frequentemente ativado por citocinas, como IL-6, que pode atuar nesse processo. Já os microRNAs (miRNAs) regulam a expressão gênica pós-transcricional degradando ou silenciando RNAs mensageiros. O presente trabalho objetivou quantificar os níveis de microRNAs miR-10a, miR-17, miR-29c e miR-31 em campo de cancerização de displasias e CCE. Adicionalmente, avaliou-se o padrão de expressão de IL-6 e STAT3 nessas lesões. Para tanto, foram coletadas amostras de 47 CCEs, 21 displasias e 21 de mucosa normal. Nos grupos CCE e DPM, foi coletada amostra adicional de mucosa perilesional, distando 1 cm da lesão. Os miRNAs foram analisados por PCR em tempo real e STAT3 e IL-6 por imunoistoquímica (IQ). Os casos de CCE eram, predominantemente, moderadamente diferenciados (91,5%), com crescimento em lençóis ou ninhos (73%) e não-ceratinizantes (52,6%). As displasias leves apresentaram-se mais frequentemente (52%), sendo que 42,8% mostravam inflamação subepitelial intensa. A idade média dos pacientes dos três grupos foi de 52±21 anos. Houve predomínio do sexo masculino no grupo CCE (67,3%) e do feminino nas displasias (57,1%). Detectou-se hiperexpressão dos miRNAs estudados em CCE e displasias, assim como nas regiões perilesionais. Identificou-se correlação positiva entre todos os miRNAs estudados no grupo CCE, enquanto na área adjacente ao tumor o aumento de miR-31 foi acompanhado de elevação nos níveis de miR-17, assim como o aumento de miR-29c foi seguido por incremento de miR-10a. Houve aumento significativo da expressão de miR-17 e miR-29c em CCE em relação a displasias. Nestas, houve correlação positiva do miR-10a com miR-29c e miR-17, ocorrendo situação semelhante entre miR-31 e miR-17. Em região perilesional de DPM, os microRNAs miR-10a e miR-29c apresentaram correlação positiva. O aumento da expressão de miR-31 foi relacionado a displasias com classificação histológica mais severa. Obteve-se alta expressão de IL-6 em 100% das amostras de CCE e DPM. STAT3 apresentou positividade para 84,6% das amostras de CCE e 88,2% de DPM. Correlação positiva entre miR-29c e miR-10a foi encontrada no CCE, nas displasias e áreas perilesionais, indicando que a hiperexpressão desses microRNAs é um evento precoce no processo de carcinogênese oral.