Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme following cyclooxygenase-2 (COX-2) for the production of prostaglandin E₂(PGE₂), and has been identified as a novel therapeutic target for cancers. From an in silico screen aimed at developing novel small molecule inhibitors of mPGES-1, a 2-aminothiazole compound PGE0001 was identified from 13 putative hits based on its ability to reduce cellular PGE₂ and minimal COX-2 inhibition in vitro. Utilizing drug design strategies based on a 4-point pharmacophore model, we also discovered a new series of compounds exhibiting superior potency without inhibiting COX-2, as exemplified by compound PGE0056. In multiple cancer cell lines, both PGE0001 and PGE0056 reduced cytokine-stimulated PGE₂ release with submicromolar EC₅₀ values, although the two compounds exhibited differential kinetics. Importantly, these compounds showed promising anti-tumor effects in xenograft mouse models. Mice injected with the compounds also had reduced PGE₂ in serum. Surprisingly, none of the compounds inhibited mPGES-1 in cell-free assays, except for MK-886, a reported mPGES-1 inhibitor. In order to determine the mechanisms of action of PGE0001 and PGE0056, the PGE₂ synthesis cascade was extensively examined. Immunoblotting analysis suggested that the PGE₂ reduction in a short time frame was not due to alteration of the protein level of enzymes involved in PGE₂ synthesis/metabolism. So far, we have excluded upstream COX-1/2, phospholipase A₂, and other PGE synthases (mPGES-2 & cytosolic PGES) as major targets for PGE0001 or PGE0056. Interestingly, these compounds were found to inhibit a number of kinases implicated in cancer, presumably due to their structural feature. Although these alternative kinase targets may not sufficiently explain the mechanisms responsible for PGE₂ reduction, inhibition of them may strengthen the therapeutic potential of our compounds. We also implemented a target pull-down approach using biotinylated derivatives of these compounds, followed by proteomic analysis to isolate targets to which these compounds bind. As a result, we identified a couple of other enzymes involved in the arachidonic acid metabolic pathway, which need to be further validated. In summary, we identified novel classes of anti-inflammatory compounds with anti-tumor activity, although the mechanisms of action remain to be clarified.

No description available.

No description available.

No description available.

Memoria para optar al Titulo Profesional de Médico Veterinario / La apoptosis o muerte celular programada es la eliminación de células que ya no son necesarias o que están dañadas genéticamente. Es controlada por una variedad de genes, muchos de los cuales presentan mutación y/o disfuncionalidad en su regulación, asociada a cáncer. Cuando esto ocurre, los pacientes presentan tumores más agresivos. La medición morfológica cuantitativa del índice apoptótico, especialmente por microscopía de luz, es difícil ya que los cambios celulares asociados son de corta duración. En la apoptosis ocurre fragmentación del ADN, la que puede ser detectada y marcada enzimáticamente mediante la técnica denominada TUNEL (terminal transferense mediaded dUTP-biotin nick end labelling). Permite detectar estados tempranos de apoptosis, incluso antes que el núcleo experimente los primeros cambios morfológicos. El Tumor Venéreo Transmisible (T.V.T), se caracteriza por presentar una fase de crecimiento progresivo muy acelerado con gran destrucción tisular local. Sin embargo, el tratamiento con sulfato de Vincristina, a diferencia de lo que ocurre en otras neoplasias, induce la regresión de la masa tumoral hasta su completa desaparición En este trabajo se seleccionaron 10 caninos adultos con TVT de ubicación genital en fase progresiva de crecimiento para inducirles regresión tumoral con Sulfato de Vincristina en dosis única de 0.03mg/Kg. Se obtuvieron muestras histológicas para estudiar el comportamiento apoptótico del tumor después del tratamiento, mediante la técnica de inmunotinción TUNEL. Se digitalizaron imágenes que fueron analizadas con un software morfométrico (Image Pro-Plus, Media Cybernetics, USA). Se observó una intensa inmunomarcación para apoptosis en tejidos de TVT en fase regresiva, involucrando muchas células que no presentaban aún cambios morfológicos asociados a apoptosis, contrastando con una inmunomarcación ocasional, de células aisladas, en la fase progresiva de crecimiento. El área promedio de células apoptóticas fue de 51.3±37.9m2 y 1396±828.6m2, por campo de 200X, para fase progresiva y regresiva, respectivamente, indicando una diferencia significativa entre ambas fases (p<0.0001). El tipo celular principal dentro de la población apoptótica, en ambas fases, correspondió a células tumorales con un 80.7% para fase progresiva y 89.4% para fase regresiva. Mientras, que los linfocitos representaban un 16.1% y 8.4%, en las fase progresiva y regresiva, respectivamente. Las epiteliales constituían sólo un 3.2% en fase progresiva y 2.2% en fase regresiva. Dentro de la población apoptótica, las células de T.V.T. mostraron un aumento significativo en fase regresiva (p<0.001). Los linfocitos disminuyeron en forma significativa en regresión (p<0.001). En cambio, la disminución registrada en las células epiteliales en fase regresiva no fue estadísticamente significativa (p ≥0.05). Este estudio indica que la Vincristina induce regresión en T.V.T. a través de apoptosis, llevando al colapso de la masa tumoral. El mecanismo por el cual la Vincristina induciría apoptosis en T.V.T. permanece aún por aclarar, sin embargo se ha demostrado, en otras neoplasias, activación de caspasas 9 y 3, sugiriendo que este tratamiento induce apoptosis por la vía mitocondrial, involucrando mecanismos asociados a la generación de radicales derivados del oxígeno y sobre expresión de gen Bcl-2

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Approximately 25% of patients with locoregional esophageal adenocarcinoma (EC) are resistant (marked by minimal tumor regression; TRG 3) to preoperative chemoradiation, including 5FU‐based and CROSS regimens. Previously, an immunohistochemistry (IHC) test that accurately identifies patients as responders (TRG 0‐2) or non‐responders (TRG 3) to neoadjuvant CTRT was developed and validated. The current study was designed to identify gene expression profile (GEP) signatures able to predict response to preoperative treatment. Methods: Formalin‐fixed, paraffin‐embedded (FFPE) tumor tissue from 24 diagnostic biopsies (14 responders, 10 non‐responders) was collected. RNA was isolated, and RT‐PCR performed to assess the expression of 96 candidate genes chosen from in silicoanalysis. Genetic signatures incorporating genes with significant expression differences in pathologically determined responders versus non‐responders were identified, and linear and non‐linear predictive modeling methods were used to assess the accuracy of the signatures for predicting treatment response. Cross validation was performed to attain corrected accuracy values. Ten‐, 18‐, and 24‐gene signatures were identified with significantly different gene expression levels in responders compared to non‐responders (p < 0.05). Functional groups represented by the signatures included DNA damage repair, extracellular matrix remodeling, and 5FU metabolism. Partial Least Squares (PLS) prediction of treatment response was compared to pathologic TRG determined by blinded pathologic reading, and resulted in an area under the curve (AUC) of 0.99 and overall accuracy of 100% for the 24‐gene signature. Corrected AUC of 0.99 and accuracy of 95% resulted from five‐fold cross validation with 20 iterations. Heatmap analysis of the 24‐gene signature separated the EC cases into two distinct clusters, the first with 93% responders and the second with 90% non‐responders. The current study identifies novel gene signatures able to accurately predict EC patient response to preoperative treatment. The GEP may allow non‐responders to avoid unnecessary toxicities associated with chemoradiation therapy.

Les glycanes présents à la surface des cellules cancéreuses sont souvent modifiés par rapport à ceux d’une cellule saine. Or ces antigènes glucidiques n’induisent pas de réponse immune efficace. La GalNAc est le premier sucre fixé lors de la O-glycosylation de type mucine et ainsi ce sucre entre dans la composition de nombreux antigènes tumoraux. Le but de notre travail était de préparer des analogues synthétiques de la GalNAc susceptibles d’être incorporés à la surface de cellules cancéreuses et dans les mucines synthétisées par les tumeurs, afin d’augmenter la réponse immune vis-à-vis des glycanes tumoraux. Nous avons synthétisé chimiquement des analogues de la GalNAc afin de les tester in vitro en tant que substrats de la voie de « sauvetage » de la GalNAc chez les mammifères et donc d’enzymes impliquées dans cette voie : une kinase (GK2) et une UDP-pyrophosphorylase (AGX1) humaines. Les meilleurs candidats ont permis la synthèse de différents UDP-sucres et une GalNAc-transférase (ppGalNAc T1) bovine a pu être utilisée in vitro pour transférer certains de ces analogues, à partir de leur forme activée en UDP-sucre, sur des peptides. Nous avons donc pu montrer que certains des analogues synthétisés étaient capables de s’intégrer dans la voie de sauvetage et d’être incorporés dans des peptides. Le pouvoir immunologique des glycoconjugués de type mucine ainsi formés a été étudié chez la souris après couplage de ces glycoprotéines à une protéine immunostimulante (KLH). D’autre part, des cellules de mammifères ont également été cultivées en présence de ces analogues afin de vérifier leur incorporation au niveau des glycoconjugués de la surface des cellules. / Glycans are often present at the cancerous cell surface in a modified form compared to healthy cells. However, these carbohydrate antigens don’t lead to an effective immune response. GalNAc is the first sugar attached to mucin type O-glycans and is thus a component of numerous tumor antigens. The aim of our work was to prepare synthetic GalNAc analogs able to be incorporated at the surface of cancer cell and into mucins synthesized by tumors in order to increase the immune response toward tumor glycans. We chemically synthesized GalNAc analogs to test them in vitro as substrates of enzymes involved in the mammalian GalNAc salvage pathway: a human galactokinase (GK2) and a human UDP-pyrophosphorylase (AGX1). The best candidates allowed the synthesis of the corresponding UDP-sugars further used to test the transfer of those analogs onto peptides using a bovine GalNAc transferase (ppGalNAc T1). We have shown that some synthetic analogs could be integrated in the GalNAc salvage pathway and O-linked to peptides. Immunological properties of the glycoconjugates thus formed were studied in mice after coupling to an immunostimulant protein (KLH). Moreover, mammalian cells were cultivated in the presence of these analogs in order to check their incorporation into glycoconjugates at the cell surface.

Platelets play a crucial role for blood hemostasis, the process that prevents bleeding. In addition, platelets have been demonstrated to promote cancer progression and cancer related complications like metastasis and thrombosis. Platelets can affect cancer related diseases either directly or by interacting with other blood cells or molecules in the circulation of individuals with cancer. The current thesis addresses the role of platelets in tumor progression and tumor-induced systemic effects of cancer, with a special focus on the effects on the vasculature. In the first paper, the role of platelets in tumor progression in histidine-rich glycoprotein (HRG)-deficient mice was addressed. We report that HRG-deficient mice show enhanced tumor growth, epithelial to mesenchymal transition (EMT) and metastasis. The enhanced platelet activity in the absence of HRG is responsible for the accelerated tumor progression. In the second paper, we demonstrate that platelet-derived PDGFB is a central player to keep the tumor vessels functional. Moreover, in a pancreatic neuroendocrine carcinoma model with PDGFB-deficient platelets, spontaneous liver metastasis was enhanced. With this finding we identify a previously unknown role of platelet derived PDGFB. In the third paper, we found that TBK1 mediates platelet-induced EMT by activation of NF-kB signaling, which suggest that TBK1 contributes to tumor invasiveness in mammary epithelial tumors. In the last paper, we report that the vascular function in organs that are neither affected by the primary tumor, nor represent metastatic sites, is impaired in mice with cancer. We show that tumor-induced formation of intravascular neutrophil extracellular traps (NETs), a fibril matrix consisting of neutrophils with externalized DNA and histones, granule proteases and platelets, are responsible for the impaired peripheral vessel function.

CTCs have emerged as a potential source of clinical significance. But with numerous isolating systems currently available, the numbers of captured CTCs vary widely. At this point, CellSearch remains the only FDA-approved system with clinical significance whereby the results could be used to monitor patients with metastatic colon, breast, or prostate cancer. However, its inability to isolate CTCs from non-high risk prostate cancer patients or CTCs that are EpCAM-negative has led to criticism. In this study, we have shown that size-based filtration successfully isolates CTCs from patients with localized and metastatic prostate cancer. We have also shown that CTCs can be successfully isolated from low and intermediate risk groups. Additionally, clusters of CTCs were preserved and isolated in all localized risk groups and metastatic patients. Furthermore, we enumerated the isolated CTCs using automated and manual methods in low risk, intermediate risk, high risk, and metastatic prostate cancer. The automated and manual counts were comparable. Moreover, the amounts of clusters and the size of clusters correlated with the status and stage of prostate cancer. / October 2016

Tumors have several important hallmarks; anomalous and heterogeneous behaviors of their vascular structures, and high angiogenesis and neovascularization. Tumor tissue presents high blood flow (F) and extraction ratio (E) of contrast molecules. Consequently there is growing interest in non invasive methods for characterizing changes in tumor vasculature. Toft's model has been extensively used in the past in order to calculate Ktrans maps which take into consideration both F and E. However, in this thesis we argue that for accurate tumor characterization we need a model able to compute both F and E in tissue plasma. This project has been developed as part of a larger project, working toward building a Clinical Decision Support System (CDSS): an interactive expert computer software, that helps doctors and other health professionals make decisions regarding patient treatment progress. Using the Gamma Capillary Transit Time (GCTT) pharmacokinetic model we calculate F and E separately in a more realistic framework; unlike other models it takes into account the heterogeneity of the tumor, which depends on parameter a-1. a-1 is the width of the distribution of the capillary transit times within a tissue voxel. In more detail, a-1 expresses the heterogeneity of tissue microcirculation and microvasculature. We studied 9 patients pathologically diagnosed with glioblastoma multiforme (GBM), a common malignant type of brain tumor. Several physiological parameters including the blood flow and extraction ratio distributions were calculated for each patient. Then we investigated if these parameters can characterize early the patients' responsiveness to current treatment; we assessed the classification potential based on the actual therapy outcome. To this end, we present a novel analysis framework which exploits the new parameter a-1 and organizes each voxel into four sub-region. Our results indicate that early characterization of response based on GCCT can be significantly improved by focusing on tumor voxels from a specific sub-region.