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El herpesvirus asociado al sarcoma de Kaposi, KSHV, es el agente etiológico del sarcoma de Kaposi. Dentro del genoma viral, el receptor viral acoplado a proteína G (vGPCR) conduce a la oncogénesis y angiogénesis; su expresión persistente y actividad es necesaria para el mantenimiento del tumor. La forma hormonal activa de la vitamina D, 1α,25(OH)2D3, presenta efectos antineoplásicos en varios tipos de tumores. El potencial anti-proliferativo y antiinflamatorio del 1α,25(OH)2D3 en células endoteliales que expresan el vGPCR ha sido explorado por nuestro grupo. En los últimos años se ha incrementado el uso de análogos sintéticos del 1α,25(OH)2D3 con menor actividad calcemica para minimizar la hipercalcemia producida por dosis altas del metabolito. En este trabajo de Tesis Doctoral se profundizó en el mecanismo de acción antineoplásico del 1α,25(OH)2D3 y su análogo menos calcemiante TX 527. Se obtuvo evidencia que ambos compuestos promueven la apoptosis a través de un desbalance entre los miembros de la familia de proteínas Bcl-2 en células vGPCR. Se observó un aumento en los niveles de expresión de la proteína pro-apoptótica BIM y disminución de A20 por un mecanismo dependiente del VDR y un pico de inactivación en la fosforilación de BAD acompañado por la formación de complejos con Bcl-2, alterando posiblemente la integridad de la mitocondria. También, se demostró que el 1α,25(OH)2D3 y TX 527 inducen autofagia a través de la inactivación del eje PI3K/Akt/mTOR y regulación de BECN1 como parte del mecanismo antineoplásico de acción. Asimismo, ambos agonistas regulan la proliferación de las células vGPCR mediante la inactivación de ERK1/2 y p38 y aumento de la expresión de las fosfatasas MKP-3 y MKP-5. Eventos regulados por la expresión del VDR, con excepción de ERK1/2, debido probablemente a una regulación no genómica de esta quinasa. El Bortezomib, inhibidor del proteasoma y de la activación de NF-ĸB, disminuyó la proliferación celular e indujo apoptosis por un mecanismo similar a los agonistas del VDR en células vGPCR. Proceso acompañado además por la activación del factor de transcripción FOXO1, disminución en la expresión génica de VEGF y aumento del inhibidor del ciclo celular, p21. Debido a las limitaciones que presentan los cultivos celulares bidimensionales, se desarrolló un método para obtener esferoides multicelulares a partir de células endoteliales y transformadas por la expresión del vGPCR. Las células vGPCR cultivadas en una superficie de baja adherencia desarrollaron esferoides de mayor tamaño y el tratamiento con 1α,25(OH)2D3 produjo cambios morfológicos con la consecuente inducción de apoptosis. A nivel molecular, se observó un aumento en la expresión génica del VDR, BIM y p21 al aumentar la dosis. Además, el 1α,25(OH)2D3 provocó un aumento en los niveles proteicos de BIM y disminuyó la activación de ERK1/2 y Akt, permitiendo concluir que las células en cultivos tridimensionales responden al agonista de manera similar a lo observado en cultivos en monocapa. Los resultados obtenidos en este trabajo de tesis contribuyen al conocimiento del mecanismo de acción antiproliferativo del 1α,25(OH)2D3 y su análogo menos calcemiante TX 527 en un modelo celular de sarcoma de Kaposi. A su vez, sustenta las bases para dar continuación a otros estudios en modelos in vivo y evaluar su potencial aplicación, sólo o en combinación con Bortezomib, en el tratamiento de esta patología. / The Kaposi's sarcoma-associated herpesvirus, KSHV, is the etiological agent of Kaposi's sarcoma. Within the viral genome, the viral G protein-coupled receptor (vGPCR) leads to oncogenesis and angiogenesis; its persistent expression and activity are necessary for tumor maintenance. The active hormonal form of vitamin D, 1α,25(OH)2D3, has antineoplastic effects in several types of tumors. The anti-proliferative and antiinflammatory potential of 1α,25(OH)2D3 in endothelial cells expressing vGPCR has been explored by our group. In recent years, the use of synthetic analogues of 1α,25(OH)2D3 with lower calcemic activity has been increased to minimize the hypercalcemia produced by high doses of the metabolite. In this Doctoral Thesis work, the antineoplastic mechanism of action of 1α,25(OH)2D3 and its less calcemic analogue TX 527 was studied. Evidence was obtained that both compounds promote apoptosis through an imbalance of Bcl-2 protein family members in vGPCR cells. An increase of pro-apoptotic BIM and a decrease of A20 protein expression levels by a VDRdependent mechanism was observed, as well as, a peak of inactivation of phosphorylated BAD accompanied by its association with Bcl-2, possibly altering mitochondria integrity. It was also demonstrated that 1α,25(OH)2D3 and TX 527 induce autophagy through the inactivation of the PI3K/Akt/mTOR axis and regulation of BECN1 as part of the antineoplastic mechanism of action. Moreover, both agonists regulate vGPCR cells proliferation by inactivating ERK1/2 and p38 and increasing MKP- 3 and MKP-5 phosphatases expression. These events were regulated by VDR expression, with the exception of ERK1/2, probably due to a non-genomic regulation of this kinase. Bortezomib, an inhibitor of proteasome and NF-ĸB activation, decreased cell proliferation and induced apoptosis by a similar mechanism to VDR agonists in vGPCR cells. Process also accompanied by the activation of the transcription factor FOXO1, decrease in VEGF gene expression and increase of the cell cycle inhibitor, p21. Due to the limitations of two-dimensional cell cultures, a method was developed to obtain multicellular spheroids from endothelial cells and transformed by the expression of vGPCR. vGPCR cells cultured on a low adhesion surface developed larger spheroids and treatment with 1α,25(OH)2D3 triggered morphological changes with consequent apoptosis induction. At molecular level, an increase in VDR, BIM and p21 gene expression was observed when dose was increased. In addition, 1α,25(OH)2D3 caused an increase in BIM protein levels and decreased the activation of ERK1/2 and Akt, allowing to conclude that cells in three-dimensional cultures respond to the agonist in a similar way to that observed in monolayer cultures. The results obtained in this thesis work contribute to the knowledge of the anti-proliferative mechanism of action of 1α,25(OH)2D3 and its less calcemic analogue TX 527 in a cellular model of Kaposi's sarcoma. In turn, it supports the bases to continue other studies in vivo models and evaluate their potential application, either alone or in combination with Bortezomib, in the treatment of this pathology.

Inflammation has been found to play a role in the development of many different tumors. However, a tumor's ability to evade immune cell recognition can be integral to its progression as well. The following works explore this complicated role with a focus on histiocytic sarcoma (HS) and breast cancer. Chapter 1 opens with a broad overview of inflammation in tumorigenesis while Chapter 2 focuses on a review and discussion of current HS literature. Our investigations into the role of inflammation specifically in HS are initiated in Chapter 3 where we explore the role of the regulatory NLR, NLRX1, in the development of HS in mice. NLRX1 is an intracellular patter recognition receptor that functions to regulate pro-inflammatory cell pathways. Our studies reveal that in carcinogen-induced HS in mice, NLRX1 acts as a tumor suppressor. Moreover, when NLRX1 is lost, tumors that develop are associated with increases in expression of genes in NF-κB and AKT pathways. Though uncommon, HS is a clinically relevant tumor in dogs. In Chapter 4, we further investigate the role of the pathways identified in Chapter 3 in canine patients. Not only were these pathways increased, but our results also revealed previously unreported differences in tumors diagnosed as HS versus those diagnosed as hemophagocytic HS. To improve the use of canine HS both as an experimental and translational model, we sought to create a murine xenograft model. In Chapter 5, we discuss the development of our model and the results of pilot studies using targeted drug therapy. The focus of Chapters 3-5 is to further explore the role of inflammation in the development of HS. However, as aforementioned, the role of inflammation in tumorigenesis is quite complicated. In Chapter 6, we aim to address the concept that the lack of inflammation through immune evasion, can also be important in tumors. Breast cancer in humans is traditionally recognized as being highly immunosuppressive. In this final chapter, we investigate the use of an attenuated strain of bacteria to treat these tumors by way of shifting the immunosuppressive tumor microenvironment to a more pro-inflammatory state. / Ph. D.

Increased resting energy expenditure has been postulated to be one of the contributory factors in the development of cancer cachexia. Body composition and resting energy expenditure were evaluated in six male and seven female normal controls in order to validate methodology. Identical methods were then applied to seven male sarcoma patients with localized disease. Only the age and sex-matched group of controls were compared to the sarcoma patient group. All patients had received no prior cancer treatment. Body composition assessment included measurement of body fat using four-site skinfold measures. measurement of total body potassium (40K) as an indicator of body cell mass. and calculation of body surface area. Resting energy expenditure was measured by indirect calorimetry in an enclosed plexiglass hood and compared to predicted resting energy expenditure as determined by the Harris and Benedict formulae. Measured resting energy expenditure per unit body surface area was significantly increased in the sarcoma group: 1610.7 +/- 369.2 (sarcoma) vs. 1290.3 +/- 74.3 kcal/m²/day (controls), p<.05. Percent body cell mass was significantly decreased in the sarcoma group: 32.6 +/- 3.8% (sarcoma) vs. 39.8 +/- 3.7% (controls), p<.05. Predicted resting energy expenditure underestimated measured values by 42.1 +/-13.0% in the sarcoma group and 29.7 +/- 5.4% in the control group. Results of this study suggest that in otherwise asymptomatic cancer patients with metabolically active tumors, such as sarcomas, increased resting energy expenditure contributes to the onset of cancer cachexia prior to any signs of overt host depletion. / M.S.

Histiocytic sarcoma (HS) is an exceptionally rare malignant neoplasm derived from dendritic cells and histiocytes, with no available effective treatment options. Akt signaling and proteasome dysfunction have been implicated in the pathogenesis of the disease, both in humans and dogs. Our work aims to investigate the importance of the Akt signaling pathway and evaluate the potential of Akt-targeted therapy in a canine model of histiocytic sarcoma. We demonstrated Akt signaling to be active in 9 out of 10 canine HS tumor samples, regardless the presence of PTEN. Moreover, the Akt signaling pathway appears to be constitutively active in DH82 cells — a cell line model of canine HS, when compared to control canine dendritic cells. Pharmacologic Akt inhibition resulted in significant decrease in Akt S473 phosphorylation, GSK-3β S9 phosphorylation, Akt activity, cell viability, increased apoptosis, and resulted in sensitization to proteasome inhibition-depended cell death in a synergistic manner. Proteasome inhibition using carfilzomib, an irreversible proteasome inhibitor, induced dose-depended/caspase-3 independent cell death, at clinically relevant drug concentrations. The therapeutic effect of Akt inhibition was validated in vivo using a DH82 xenograft murine model. Akt inhibition lead to reduced tumor growth, prolonged overall survival, and ameliorated splenomegaly, but not affected the lung metastasis. Moreover, the therapeutic effect of Akt inhibition was potentiated in combination with carfilzomib. In conclusion, targeting Akt signaling may represent an attractive potential therapeutic target for the HS. Future studies are required to examine the clinical efficacy of Akt-targeted therapy in dogs with HS using novel selective Akt inhibitors. / Ph. D. / Histiocytic sarcoma (HS) is an exceptionally rare cancer of the immune system, with no effective treatment options available. Canine histiocytic sarcoma (cHS) is an aggressive tumor of the same cellular lineage, identified at increased relative frequency in specific dog breeds, with significant translational value. Akt signaling and proteasome dysfunction have been implicated in the pathogenesis of the disease, both in humans and dogs. Our study aims to investigate the importance of the Akt signaling pathway in the dog model of the disease and evaluate the potential of Akt-targeted therapy in a translational of histiocytic sarcoma. The work presented here demonstrates that Akt signaling appears aberrantly and constitutively activated in the canine model of HS. Importantly, Akt inhibition significantly reduced the tumor growth and prolonged the overall survival of the experimental animals. Moreover, Akt inhibition potentiated the anti-cancer activities of other anticancer drugs. Collectively, these findings provide an attractive therapeutic approach for the treatment of HS.

Yes / Background: Soft tissue sarcomas are heterogeneous and a major complication in their management is that the existing classification scheme is not definitive and is still evolving. Leiomyosarcomas, a major histologic category of soft tissue sarcomas, are malignant tumours displaying smooth muscle differentiation. Although defined as a single group, they exhibit a wide range of clinical behaviour. We aimed to carry out molecular classification to identify new molecular subgroups with clinical relevance. Methods: We used gene expression profiling on 20 extra-uterine leiomyosarcomas and cross-study analyses for molecular classification of leiomyosarcomas. Clinical significance of the subgroupings was investigated. Results: We have identified two distinct molecular subgroups of leiomyosarcomas. One group was characterised by high expression of 26 genes that included many genes from the sub-classification gene cluster proposed by Nielsen et al. These sub-classification genes include genes that have importance structurally, as well as in cell signalling. Notably, we found a statistically significant association of the subgroupings with tumour grade. Further refinement led to a group of 15 genes that could recapitulate the tumour subgroupings in our data set and in a second independent sarcoma set. Remarkably, cross-study analyses suggested that these molecular subgroups could be found in four independent data sets, providing strong support for their existence. Conclusions: Our study strongly supported the existence of distinct leiomyosarcoma molecular subgroups, which have clinical association with tumour grade. Our findings will aid in advancing the classification of leiomyosarcomas and lead to more individualised and better management of the disease. / Alexander Boag Sarcoma Fund.

D.Litt. et Phil. ( Psychology) / A literature survey has confirmed that the growing number of cancer survivors in general are experiencing both physical as well as psychological and social adaptation difficulties following their cancer treatment. This is no different for patients diagnosed with Osteosarcoma and Ewing sarcoma (bone sarcomas) during adolescence. At present there is no formal psychosocial intervention program available to the patients presenting with Ewing and Osteosarcomas. Because of the multicultural nature of the patients as well as the fact that many childhood and adolescent cancers do not include major surgery, as is the case with bone sarcomas, the direct applicability of generalized psychosocial intervention programs for adolescents with cancer other than bone sarcoma is doubtful ...

A ressecção ampla do trajeto de biópsia junto ao tumor faz parte da técnica cirúrgica oncológica para evitar os riscos de implante de células tumorais e recidiva local e/ou sistêmica da doença. O objetivo deste estudo foi verificar se havia risco de contaminação por células neoplásicas no trajeto da biópsia. Realizou-se um estudo prospectivo dos trajetos de biópsias de pacientes operados por tumores musculoesqueléticos malignos no Instituto de Ortopedia e Traumatologia da Faculdade de Medicina da Universidade de São Paulo no período de abril de 2006 a abril de 2007. Foram estudados por histopatologia 25 casos. Houve implante de células neoplásicas em 32% dos trajetos. Quando foram correlacionados os resultados dos estudos dos trajetos e o uso de quimioterapia neo-adjuvante (p=0,19) não houve significância, mostrando que mesmo com esse tratamento o risco de implante existe. As alterações histológicas mais comuns foram: fibrose acentuada, componente inflamatório leve e neovascularização acentuada. Sugerimos a ressecção tradicional oncológica do trajeto junto com a peça / Wide resection of biopsy tracts to tumors forms part of oncological surgical techniques because of the risk of tumor cell implantation and local and/or systemic disease recurrence. The aim of this study was to investigate the risk of seeding by neoplastic cells along biopsy tracts. This was a prospective histopathological study on biopsy tracts in 25 patients who underwent operations due to malignant musculoskeletal tumors, at the Institute of Orthopedics and Traumatology, School of Medicine of the University of São Paulo, between April 2006 and April 2007. Neoplastic cells were found implanted in 32% of the tracts. Correlation of the results from studying the tracts with the use of neoadjuvant chemotherapy did not present significant findings. This shows that, even with chemotherapy, the risk of implantation exists. The most common histological abnormalities in positive cases were classified as severe fibrosis, mild inflammatory component and severe neovascularization. We suggest that oncological resection of the tract should be carried out together with excision of the specimen

O Sarcoma de Ewing (ES) é um raro tumor de ossos e tecidos moles com uma característica translocação cromossomal, a fusão EWS/FLI-1, que atua sobre diversos processos oncogênicos. O desenvolvimento da resistência à quimioterapia é comum no tumor e continua como uma das principais causas na falha do tratamento. O objetivo desse estudo foi avaliar a expressão de genes após a indução de resistência em linhagens celulares de ES. Foi selecionado um conjunto de genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 e SMARCB1) a partir da mineração da literatura em resistência tumoral para duas drogas utilizadas na terapia de ES, doxorrubicina e vincristina. Descrevemos a expressão de cada gene selecionado antes e após as linhagens SK-ES-1 serem submetidas a um protocolo de indução de resistência para ambos os fármacos, que obteve êxito ao induzir as células à resistência. A expressão relativa dos níveis de mRNA foi avaliada e foi encontrada em maior expressão para os genes SMARCA4, SMARCB1 e POLDIP2, e em menor expressão para os genes TUBA1A e CCAR1, quando comparadas às linhagens de controle não-resistentes de cada quimioterápico. Os resultados sugerem o envolvimento de mecanismos de reparo de dano ao DNA, remodelamento de cromatina via SWI/SNF, atividade de microtúbulos e atividade spliceossomal nos processos de resistência quimioterápica em ES. / Ewing Sarcoma (ES) is a rare bone and soft tissue tumor with a characteristic chromosomal translocation, the fusion protein EWS/FLI-1, that drives several oncogenic processes. The development of resistance to chemotherapy is common and remains as the main cause of treatment failure. The goal of this study was to evaluate the expression of selected genes in ES cell lines after induction of resistance. A set of genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 and SMARCB1) was data mined from tumoral resistance literature for two drugs used in ES therapy, doxorubicin and vincristine. We describe the expression of each selected gene before and after SK-ES-1 cell lines were exposed to a drug resistance inducing protocol for doxorubicin and vincristine. Cell lines were successfully induced to be resistant to doxorubicin and vincristine. The relative mRNA expression levels were upregulated for genes SMARCA4, SMARCB1 and POLDIP2 and downregulated for genes TUBA1A and CCAR1, when comparing resistant and non-resistant ES cell lines for each drug. The results suggest involvement of repair pathways, SWI/SNF chromatin remodeling, microtubule and spliceosomal activity processes in drug resistance mechanisms in ES.

Objetivo: Este estudo foi realizado com objetivo de traduzir para o português do Brasil e validar a ferramenta Musculoskeletal Tumor Society Rating Scale (MSTS) para avaliação da função em pacientes com sarcomas ósseos dos membros inferiores. Método: A ferramenta MSTS foi traduzida da língua inglesa para o português do Brasil. As versões traduzidas foram sintetizadas em uma versão de consenso que foi retrotraduzida para o inglês e analisada por um comitê multidisciplinar para posterior aplicação da ferramenta. O questionário foi aplicado em 67 pacientes operados por tumores musculoesqueléticos ósseos malignos de membros inferiores com cirurgia preservadora de membro ou amputação. Este grupo também preencheu uma versão validada em Português do Toronto Extremity Salvage Score (TESS). Foram realizadas as análises de confiabilidade do teste e reteste, confiabilidade interobservador, consistência interna, validação do instrumento e correlação com o TESS. Resultados: A confiabilidade do teste e reteste e interobservadores foi feita através da análise dos coeficientes de correlação intraclasse (ICC) com valores de 0,92 e 0,98 respectivamente (intervalo de confiança de 95%). Na análise da consistência interna, o valor do coeficiente alfa de Cronbach foi de 0,84. A correlação entre as ferramentas MSTS e TESS foi moderada. Conclusão: A ferramenta MSTS foi traduzida e validada para o Português do Brasil e mostrou ser confiável para aplicação em pacientes com sarcomas ósseos de membros inferiores. Isto possibilitará a avaliação funcional dos pacientes brasileiros e comparação com resultados de estudos multicêntricos / Objective: This study was designed to translate and validate the Brazilian version of the Musculoskeletal Tumor Society Rating Scale (MSTS) for functional evaluation in patients with lower extremity bone sarcomas. Method: The MSTS was translated from English into Brazilian Portuguese. Translations were synthesized, translated back into English and reviewed by a multidisciplinary committee for further implementation. The questionnaire was administered to 67 patients treated for malignant lower extremity bone tumors who were submitted to limb salvage surgery or amputation. They also completed a Brazilian version of the Toronto Extremity Salvage Score (TESS). Scale reliability and validity were evaluated. Results: Testretest reliability and interobserver analysis were performed using the intraclass correlation coefficients (ICC) with values of 0,92 and 0,98 respectively (confidence interval 95%). Cronbach\'s alpha was used to estimate internal consistency (0,84). There was mild correlation between MSTS and TESS. Conclusion: The Brazilian version of the MSTS was translated and validated. It is a reliable tool to assess functional outcome in patients with lower extremity bone sarcomas. It can be used for functional evaluation of Brazilian patients and cross-cultural comparisons