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The cancer stem cell hypothesis has recently re-emerged as a compelling paradigm for the development and progression of neoplastic disease. The hypothesis proposes that a specific subset of “cancer stem cells” (CSC), believed to share many features with normal stem cells, is exclusively responsible for maintaining tumour growth and driving progression. If the CSC hypothesis applies, it may require re-evaluation of the clinical approach to neoplasia. Spontaneous cancer in the domestic dog represents a significant welfare problem, with dogs developing many tumours strongly reminiscent of those affecting humans. This study sought to investigate whether cells with characteristics of CSC are identifiable in canine cancer. Assays to identify, isolate and characterise CSC were adapted to the canine system, and cancer cell lines and spontaneous tumours of diverse origin evaluated for the presence of candidate populations. Whilst analysis of surface expression patterns did not identify specific subpopulations within canine cancer cell lines, these were detectable in cells derived directly from primary tumours. Assays for stem cellassociated drug resistance mechanisms could also be used to identify subsets of putative canine CSC. Formation of “tumourspheres” by canine cancer cell lines was found to be highly density-dependent, so a potentially unreliable method of isolating CSC. Expression of the cell surface glycoprotein CD44 was associated with cellular proliferation status, although it may not represent a stable canine CSC marker. The NFκB survival pathway, associated with apoptosis resistance of some putative CSC, was constitutively active in canine cancer cell lines; suppression using specific inhibitors could reduce cell viability, indicating that this may represent a rational therapeutic target. Overall, these studies demonstrated that CSC assays may be adapted to the canine model system, although they require rigorous interrogation to distinguish apparent CSC attributes from basic biological properties. Cell lines have provided a stable background upon which to optimise assays, but appear less likely to demonstrate discrete CSC subpopulations. Putative CSC subsets may be more readily identifiable within heterogeneous primary tumour cells. The application of some of these adapted assays within a clinical setting may enable further characterisation of individual patients’ tumours, and inform therapeutic regimes for improved treatment outcomes.

O meduloblastoma é o tumor cerebral maligno mais comum em crianças de zero a quatro anos e uma das principais causas de morbidade e mortalidade infantil. Diversos estudos têm suportado a hipótese de que a ativação de genes tipicamente expressos em células-tronco confere características mais primitivas e agressivas a células tumorais, frequentemente associadas a prognóstico clínico desfavorável. Nesse contexto, tanto fatores proteicos quanto miRNAs poderiam estimular uma reprogramação em células cancerosas, induzindo um fenótipo semelhantes ao de células-tronco. Estudos recentes apontam o envolvimento do miR-367 na reprogramação de fibroblastos ao estado pluripotente e sua expressão aberrante foi correlacionada com prognóstico desfavorável em alguns tipos de câncer. Neste trabalho, verificou-se um possível papel funcional do miR-367 na agressividade de meduloblastoma. Células de meduloblastoma de quatro diferentes linhagens, Daoy, D283-Med, CHLA-01-Med e USP-13-Med apresentaram níveis baixos de expressão de pri-miR-367 e miR-367 maduro, em relação aos níveis encontrados em células-tronco embrionárias humanas. Uma superexpressão transiente do miR-367 em células das linhagens CHLA-01-Med e USP-13-Med resultaram em uma redução significativa dos níveis proteicos de RYR3, bem como dos transcritos preditos de ITGAV e RAB23, respectivos alvos do miR-367, envolvidos em câncer. Além disso, a superexpressão de miR-367 aumentou significativamente a proliferação celular, indicada pela cinética de crescimento in vitro e pela maior porcentagem de células presentes nas fases S+G2/M do ciclo celular. Embora a sensibilidade ao tratamento com cisplatina não tenha sido alterada após superexpressão de miR-367, a capacidade de geração de neuroesferas in vitro foi significativamente aumentada. Este último resultado é interessante do ponto de vista clínico, uma vez que a capacidade de geração de neuroesferas está significativamente correlacionada com menor sobrevida de pacientes com meduloblastoma. Portanto, esses achados sugerem uma função pró-oncogênica ao miR-367, a qual pode afetar a agressividade de meduloblastoma por meio de efeitos positivos sobre a proliferação celular e propriedades de células-tronco neurais / Medulloblastoma is the most common malignant brain tumor in children aged four and younger, and is the leading cause of infant morbidity and mortality. Several studies have reported the activation of stem cell genes leading to more primitive and aggressive characteristics in tumor cell often associated with unfavorable clinical prognosis. Cell reprogramming, stimulated by tumor microenvironment factors, might induce tumor stem cells phenotype. Recent researches suggest an involvement of miR-367 in fibroblasts reprogramming into pluripotent state, as well as a correlation with poor prognosis in some cancers. In this study, we observed a possible functional role of miR-367 in medulloblastoma aggressiveness. Four different medulloblastoma cell lines, Daoy, D283-Med, CHLA-01-Med and USP-13-Med showed low rates of pri-miR-367 and mature miR-367 expression. Overexpression of miR-367 down-regulated the protein levels of its target RYR3 and of two bioinformatically predicted transcript targets encoding ITGAV and RAB23, which are involved in cancer in CHLA-01-Med and USP-13 Med cell lines. Furthermore, transfection with the miRNA mimic significantly increased cell proliferation and the percentage of cells observed in S + G2 / M phase of the cell cycle. Although the sensitivity to cisplatin treatment was not changed after overexpression of miR-367, the ability to generate neurospheres in vitro was significantly increased. This last result can be related to clinical ones because cells from medulloblastoma patients with low survival show great ability to generate neurospheres. In sum, these findings suggest a pro-oncogenic role to miR-367, which can affect medulloblastoma aggressiveness by cell proliferation and neural stem cells positive modifications

Ampliar nosso conhecimento sobre a biologia do carcinoma espinocelular bucal é fundamental para o desenvolvimento de novas estratégias terapêuticas e para melhorar a sobrevida dos pacientes acometidos por essa patologia. Para tanto, compreender as contribuições do microambiente tumoral à carcinogênese é muito importante. Um característica importante a ser avaliada do microambiente tumoral é a acidificação do meio extracelular. Considerando que o pH ácido tumoral está relacionado à maior agressividade da lesão, o objetivo deste projeto é estudar os efeitos de um microambiente ácido na biologia de células de carcinoma espinocelular bucal. Para tanto, foram realizadas duas revisões da literatura, nas quais foram avaliados os seguintes temas: influência da acidez extracelular na invasão e migração; e os mecanismos moleculares envolvidos na resistência ao tratamento quimioterápico, induzida pela acidez do microambiente tumoral. Tais revisões embasaram a construção do terceiro artigo desta tese, o qual se propôs a comparar o comportamento de células linhagens de carcinoma espinocelular bucal (SCC-4 e SCC-9) e queratinócitos (HaCat) cultivadas em meio de cultura de pH 6.8 com células mantidas em meio neutro pH 7.4. As células foram expostas de forma contínua ou intermitente ao pH 6.8 e a adaptação das células foi avaliada pelo ensaio clonogênico. Além disso, as células foram avaliadas quanto à sua capacidade migratória pelo ensaio de cicatrização de feridas e de time lapse. A expressão gênica relacionada à indiferenciação e pluripotência foi investigada por PCR em tempo real com os marcadores Bmi-1 e CD44, assim como pelo ensaio de orosferas. A resistência desses grupos de células ao tratamento anti-câncer foi avaliada pelo ensaio de viabilidade celular da sulforodamina B após o tratamento com Cisplatina. Para a análise estatística, inicialmente foi realizada a distribuição dos dados, seguido da comparação estatística dos grupos utilizando, para distribuição normal, os testes ANOVA e ANOVA de duas vias. Toda a análise foi realizada no programa GraphPad Prism 5.0 e o nível de significância considerado foi de p< 0.05.Observamos que ambas as linhagens mudaram sua morfologia para um aspecto mesenquimal. Ao avaliar o perfil migratório observou-se que as células SCC-9 apresentaram maior capacidade de migração após a exposição ao pH6.8. O aumento da migração celular pode ser causado pela indução da transição epitélio-mesênquima, visto que observamos o aumento da expressão de N-caderina (SCC-4:p<0.05) concomitante à diminuição de E-caderina (SCC-4: p<0.05). A exposição à acidez também provocou, em ambas as linhagens, aumento da capacidade de formar orosferas em placa de baixa aderência, denotando um fenótipo pluripotente (SCC-4: p=0.007/ SCC-9: p= 0.1202). Tal resultado foi reforçado com aumento da expressão gênica do marcador de célula-tronco tumoral CD44 (p= 0.0325). na linhagem SCC-4. No entanto, observamos diminuição da expressão de Bmi-1(p=0.0572) em relação ao controle. A resistência à Cisplatina aumentou nos casos de exposição contínua à acidez (SCC-4: p<0,05). O recondicionamento em meio neutro reverteu a sensibilidade celular (SCC-4: p>0,05). Concluímos que a acidez extracelular no carcinoma espinocelular bucal aumenta a capacidade de migração, induz o fenótipo de células tronco-tumorais e aumenta a resistência a quimioterápicos. / To expand our knowledge about the biology of oral squamous cell carcinoma is crucial for the development of new therapeutic strategies and to improve the survival of the patients affected by this pathology. Therefore, understanding the contributions of the tumor microenvironment to carcinogenesis is very important. An important feature to be evaluated of the tumor microenvironment is the acidification of the extracellular medium. Considering that acidic pH is related to the greater aggressiveness of the tumor, the aim of this study is to analyze the effects of an acidic microenvironment on the biology of oral squamous cell carcinoma cells. We realized two reviews of the literature, in which the following themes were evaluated: the influence of extracellular acidity on the invasion and migration; and the molecular mechanisms involved in the resistance to chemotherapeutic treatment, induced by the acidity of the tumor microenvironment. These revisions helped to construct the third article of this thesis, which proposed to compare the behavior of squamous cell carcinoma lines (SCC-4 and SCC-9) and keratinocytes (HaCat), cultured under pH 6.8 was compared to cells maintained at pH 7.4. For the statistical analysis, the data distribution was initially performed, followed by the statistical comparison of the groups using, for normal distribution, ANOVA and ANOVA two-way tests. All analysis was performed in the GraphPad Prism 5.0 program and the level of significance considered was p <0.05.After continuous or intermittent exposure to pH 6.8, cell adaptation was assessed by the clonogenic assay. In addition, the migratory capacity of the cells was evaluated by the wound healing and time-lapse assays. The gene expression related to undifferentiation and pluripotency was assessed by real-time PCR analysis of the Bmi-1 and CD44 markers, as well as by the orosphere assay. The resistance of these cell groups to anti-cancer treatment was assessed by the sulforhodamine B cell viability assay after treatment with Cisplatin. We observed that both cell lines changed their morphology to a mesenchymal aspect. When assessed the migratory profile, it was observed that SCC-9 cells showed higher migration capacity after exposure to pH6.8. Increased cell migration may be caused by the induction of the epithelial-mesenchymal transition, as we observed increased N-cadherin (SCC-4:p<0.05) expression concomitant with decreased E-cadherin (SCC-4: p<0.05). Exposure to acidity also led to increased ability to form orospheres on low-attachment dishes, denoting a pluripotent phenotype in both strains (SCC-4: p=0.007/ SCC-9: p= 0.1202). This result was reinforced by increased expression of the CD44 (p= 0.0325) tumor cell marker in the SCC- 4 cells. However, we observed a decrease in the expression of Bmi-1(p=0.0572) in relation to the control. Resistance to Cisplatin increased when cells were continuously exposed to acidity(SCC-4: p<0,05). Neutral reconditioning reversed cell sensitivity (SCC-4: p>0,05). We conclude that extracellular acidity in oral squamous cell carcinoma increases the migration capacity, induces the cancer stem cell phenotype and increases resistance to chemotherapy.

Breast cancer is the most common cancer in women and unspayed female dogs. The Epithelial to Mesenchymal Transition (EMT) is a process involved in embryogenesis, carcinogenesis, and metastasis. The Transforming Growth Factor- Beta (TGF-β) pathway and its associated transcription factors are crucial for EMT induction, during which epithelial cells lose their defining characteristics and acquire mesenchymal properties. EMT has been implicated as a driver of metastasis as it allows cells to migrate and invade different organs. Recent evidence indicates that cancer stem cells are required to establish metastatic tumours at distant sites, and that EMT may promote development of cancer cells with stem-cell characteristics, thus, the EMT pathway may be an important molecular determinant of tumour metastasis. The main objective of this project was to characterise TGF-β-induced EMT in breast cancer models. EMT was induced by TGF-β in human, canine and feline breast cancer cell lines, and confirmed by morphological changes and molecular changes at the protein level by Western blot analysis. Changes at the mRNA level were confirmed in human and canine mammary carcinoma cell lines by qRT-PCR; migratory properties were assessed by invasion assays in vitro in feline and canine mammary carcinoma cells. Importantly, we observed that feline and canine mammary carcinoma cells stimulated by TGF-β acquired stem cell characteristics including sphere-forming ability, self-renewal, and resistance to apoptosis, and also enhanced migration potential. Canine cells showed resistance to chemotherapeutic drugs after TGF-β stimulation. These data suggests a link between EMT and cancer stem-cells. Moreover, global changes in microRNA expression were mapped during TGF-β-induced EMT of canine mammary carcinoma cells. This gave significant insight into the regulation of EMT in canine cancer cells and identified several potential targets, which require further investigation. During EMT cells acquire migratory properties and cancer stem-cell characteristics, suggesting that EMT and the stem-cell phenotype are closely related during cell migration and metastasis, therefore making the TGF-β pathway a potential target for the development of novel therapies against cancer and its progression.

O meduloblastoma é o tumor cerebral maligno mais comum em crianças de zero a quatro anos e uma das principais causas de morbidade e mortalidade infantil. Diversos estudos têm suportado a hipótese de que a ativação de genes tipicamente expressos em células-tronco confere características mais primitivas e agressivas a células tumorais, frequentemente associadas a prognóstico clínico desfavorável. Nesse contexto, tanto fatores proteicos quanto miRNAs poderiam estimular uma reprogramação em células cancerosas, induzindo um fenótipo semelhantes ao de células-tronco. Estudos recentes apontam o envolvimento do miR-367 na reprogramação de fibroblastos ao estado pluripotente e sua expressão aberrante foi correlacionada com prognóstico desfavorável em alguns tipos de câncer. Neste trabalho, verificou-se um possível papel funcional do miR-367 na agressividade de meduloblastoma. Células de meduloblastoma de quatro diferentes linhagens, Daoy, D283-Med, CHLA-01-Med e USP-13-Med apresentaram níveis baixos de expressão de pri-miR-367 e miR-367 maduro, em relação aos níveis encontrados em células-tronco embrionárias humanas. Uma superexpressão transiente do miR-367 em células das linhagens CHLA-01-Med e USP-13-Med resultaram em uma redução significativa dos níveis proteicos de RYR3, bem como dos transcritos preditos de ITGAV e RAB23, respectivos alvos do miR-367, envolvidos em câncer. Além disso, a superexpressão de miR-367 aumentou significativamente a proliferação celular, indicada pela cinética de crescimento in vitro e pela maior porcentagem de células presentes nas fases S+G2/M do ciclo celular. Embora a sensibilidade ao tratamento com cisplatina não tenha sido alterada após superexpressão de miR-367, a capacidade de geração de neuroesferas in vitro foi significativamente aumentada. Este último resultado é interessante do ponto de vista clínico, uma vez que a capacidade de geração de neuroesferas está significativamente correlacionada com menor sobrevida de pacientes com meduloblastoma. Portanto, esses achados sugerem uma função pró-oncogênica ao miR-367, a qual pode afetar a agressividade de meduloblastoma por meio de efeitos positivos sobre a proliferação celular e propriedades de células-tronco neurais / Medulloblastoma is the most common malignant brain tumor in children aged four and younger, and is the leading cause of infant morbidity and mortality. Several studies have reported the activation of stem cell genes leading to more primitive and aggressive characteristics in tumor cell often associated with unfavorable clinical prognosis. Cell reprogramming, stimulated by tumor microenvironment factors, might induce tumor stem cells phenotype. Recent researches suggest an involvement of miR-367 in fibroblasts reprogramming into pluripotent state, as well as a correlation with poor prognosis in some cancers. In this study, we observed a possible functional role of miR-367 in medulloblastoma aggressiveness. Four different medulloblastoma cell lines, Daoy, D283-Med, CHLA-01-Med and USP-13-Med showed low rates of pri-miR-367 and mature miR-367 expression. Overexpression of miR-367 down-regulated the protein levels of its target RYR3 and of two bioinformatically predicted transcript targets encoding ITGAV and RAB23, which are involved in cancer in CHLA-01-Med and USP-13 Med cell lines. Furthermore, transfection with the miRNA mimic significantly increased cell proliferation and the percentage of cells observed in S + G2 / M phase of the cell cycle. Although the sensitivity to cisplatin treatment was not changed after overexpression of miR-367, the ability to generate neurospheres in vitro was significantly increased. This last result can be related to clinical ones because cells from medulloblastoma patients with low survival show great ability to generate neurospheres. In sum, these findings suggest a pro-oncogenic role to miR-367, which can affect medulloblastoma aggressiveness by cell proliferation and neural stem cells positive modifications

<p>Malignant gliomas are highly lethal cancers characterized by florid angiogenesis. Glioma stem cells (GSCs), enriched through CD133 (Prominin1) selection, are highly tumorigenic and therapy resistance. However, the mechanism through which GSCs promote tumor growth was largely unknown. As we noticed that tumors derived from GSCs contain widespread tumor angiogenesis, necrosis, and hemorrhage, we examined thepotential of GSCs to support tumor angiogenesis. We measured the expression of a panel of angiogenic factors secreted by GSCs. In comparison with matched non-GSC populations, GSCs consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, GSC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-GSC glioma cell-conditioned medium. The proangiogenic effects of GSCs on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from GSCs but limited efficacy against xenografts derived from a matched non-GSC population. As hypoxia is a key regulator of angiogenesis, I further examined hypoxic responses in GSCs to determine the molecular mechanisms underlying their angiogenic drive. I demonstrated that multiple hypoxia response genes, including the hypoxia-inducible factors (HIFs)-1a and -2a(EPAS-1) were differentially expressed in GSCs in comparison to non-stem glioma cells and normal neural progenitors. GSCs preferentially induced HIF2a; and HIF2a-regulated genes under hypoxia in comparison to non-stem glioma cells. In contrast, neural progenitor/stem cells did not induce HIF2a in response to hypoxia suggesting that the HIF2a hypoxic response is not a general stem cell response. Targeting HIF1a or HIF2a in GSCs using short hairpin RNA (shRNA) inhibited neurosphere formation efficiency, indicating a requirement for HIFs in cancer stem cell self-renewal. HIF1a and HIF2a were also necessary for VEGF expression in GSCs, but HIF2a was not required in matched non-stem glioma cells. In vivo experiments determined that knockdown of HIFs significantly attenuated the tumorigenic capacity of GSCs and increased survival of immunocompromised mice. Together, our work provides the first evidence that that GSCs can be a crucial source of key angiogenic factors in cancers due to their differential hypoxia responses. It also suggests that anti-angiogenic therapies can be designed to target GSC-specific molecular mechanisms of neoangiogenesis, including the expression and/or activity of HIF2a.</p> / Dissertation

<p>Human cancer is a leading cause of morbidity and mortality in the developed world. Contrary to the classical model in which tumors are homogeneously composed of malignant cells, accumulating evidence suggests that subpopulations of highly malignant cells play a dominant role in tumor initiation and growth. These cells have the capacity for prolonged self-renewal and they efficiently generate tumors that phenotypically resemble the parental tumor in transplantation assays. Such characteristics are reminiscent of normal stem cells, and these potently tumorigenic cells have therefore been called cancer stem cells (CSCs). Importantly, studies have shown that CSCs are central mediators of therapeutic resistance, tumor angiogenesis, and metastatic or invasive potential. In the case of malignant glioma, poor patient survival and the paucity of effective therapeutic advances have been attributed to inherent CSC growth potential and treatment resistance, respectively. For this reason, there is great interest in elucidating the molecular features of CSCs, with the ultimate hope of developing CSC-directed therapies.</p><p>Given the overlap between the highly malignant characteristics exhibited by CSCs and those promoted by the PI3K/AKT pathway, we hypothesized that AKT activity within CSCs could represent a reasonable therapeutic target for CSC-directed therapies. Indeed, a pharmacological inhibitor of AKT preferentially targeted glioma CSCs versus non-CSCs and was associated with increased apoptosis and impaired tumorigenesis. These data suggest that interventions targeting AKT could effectively target glioma CSCs. </p><p>Quite distinct from the PI3K/AKT pathway, we hypothesized that the pro-survival and pro-growth features of nitric oxide (NO) might also operate in glioma CSCs. Our experiments found that glioma CSCs produced more NO than non-CSCs, which is attributed to inducible nitric oxide synthase (iNOS) expression and activity within the CSCs. Interference with iNOS activity or expression, as well as selective NO consumption, attenuated CSC growth and tumorigenicity. The mechanism behind iNOS-mediated survival appears to involve, at least in part, suppression of the cell cycle inhibitor CDA1. iNOS inhibition decreased glioma growth in murine xenografts and human expression studies demonstrate an inverse correlation between iNOS expression and patient survival.</p><p>To more fully evaluate the biological effects of NO in CSCs, we designed a novel strategy to consume NO within mammalian cells through heterologous expression of E. coli flavohemoglobin (FlavoHb). This enzyme is a highly specific NO dioxygenase which converts NO to inert nitrate several orders of magnitude faster than iNOS synthesizes NO. Expression of FlavoHb in mammalian cells is therefore a novel and functional tool to interrogate the role of NO in cellular stress and signaling. </p><p>In summary, this doctoral thesis focuses on several molecular characteristics that define malignant CSCs and describes a novel strategy for studying NO, which is one of the CSC-specific molecular effectors.</p> / Dissertation

<p>Ovarian cancer is the fifth leading cause of cancer-related death in the United States and the most lethal gynecologic malignancy. Patients with ovarian cancer are generally diagnosed at stage III or IV, when ascites fluid becomes a common symptom. The volume of ascites positively correlates with the extent of ovarian cancer metastasis and negatively with prognosis; however, the mechanisms explaining their effect are unknown. </p><p>We hypothesize that ascites enriches for cancer stem-like cells. Our present study demonstrates that mice injected with ID8 cells, a murine epithelial ovarian cancer line, have remarkably shortened survival, when injected together with ascites supernatant derived from tumor-bearing mice. Moreover, compared to their counterparts cultured in regular medium, ID8 cells cultured in ascites fluid, or isolated directly from ascites, show an increased expression of stem cell markers Oct4 and CD133. These cells also exhibit enhanced self-renewing ability in sphere assay, suggesting that ascites enriches for stem-like cells. </p><p>Furthermore, we demonstrate that ascites enriches for cells expressing cell surface GRP78, a stress-inducible endoplasmic reticulum chaperone which also appears on the plasma membrane (memGRP78) of aggressive cancers. MemGRP78 + cells correlate with stem cell properties of self-renewal and tumor initiation, suggesting GRP78 is a novel stem cell marker. Importantly, antibodies against the COOH terminal domain of GRP78 significantly reduce the self-renewing ability of murine and human ovarian cancer cells pre-incubated with ascites.</p><p>In conclusion, our study demonstrates that ascites enriches for stem-like cells in ovarian cancer cell lines. Furthermore, the inhibitory effect of antibodies against the COOH terminal domain of GRP78 suggests that memGRP78 is a logical therapeutic target for ovarian cancer.</p> / Dissertation

Objective: MS-275, a phase trialed histone deacetylase inhibitor will be characterized for its ability reduce neuroblastoma (NB) viability and to target the tumor stem cell (TSC) population in neuroblastoma. Methods: Ability of MS-275 to reduce NB growth is characterized using a tumorigenic NB N-type cell line that has high differentiation potential. TSC enriched side population from NB and a reference teratocarcinoma cell line was analyzed as a model of TSC. The potential of MS-275 to modulate functional characteristics and markers of TSC was also investigated. Results: MS-275 induces a G1 cell cycle arrest, the intrinsic apoptosis pathway in NB and can potentially differentiate NB into a more terminal phenotype. NB TSC-like population is reduced following MS-275 treatment by the targeting of their self-renewal and drug pumping ability. Conclusions: By targeting both the NB and its TSC population, MS-275 has therapeutic potential for neuroblastoma. This warrants further in-vivo investigations.