TBX2, a member of the T-box family of transcription factors, plays important roles in embryonic development. Aberrant expression of TBX2 is observed in many cancers, and serves as an oncogene to maintain tumor cell proliferative and malignant properties. We found that TBX2 was expressed in both embryonic myoblasts and adult proliferative satellite cells, but was quickly down regulated during muscle differentiation in mouse models, which suggests an important function of TBX2 in the early myogenesis. Using molecular and cellular biology approaches we showed that TBX2 forms complex with myogenin and MyoD, and then recruits HDAC1 to muscle-specific promoters to repress the myogenin and MyoD dependent differentiation of myoblasts. In rhabdomyosarcoma (RMS), which is typically referred to as a muscle derived cancer, we found TBX2 was over expressed in both major subtypes of RMS. The deregulated TBX2 repressed the expression of cell cycle regulators, such as p21 and p14/p19, and the tumor suppressor PTEN in RMS tumor cells. Knock down of TBX2 significantly decreased the proliferation rate of RMS cells. We also found that loss of TBX2 significantly inhibited tumorigenesis of RMS cells by decreasing cell proliferation, mobility, migration, anchorage-independent growth and xenograft formation. To determine why TBX2 was deregulated in RMS cells, we performed cellular biological experiments to understand how TBX2 is regulated by cell signaling pathways and growth factors in both normal muscle myoblasts and RMS tumor cells. In normal murine myoblasts and primary murine ARMS tumor cells TBX2 was up regulated by FGF-2 treatment, but in primary murine ERMS cells TBX2 expression showed no response to FGF-2 stimulation. In human RMS cell lines a modest up regulation of TBX2 was detected by treatment of FGF-2. RMS cells constitutively express PAX3 and PAX7 which are expressed and function in myogenic precursors, but are quickly degraded in myoblasts and during myogenesis. We found that TBX2 was a downstream target of PAX3 in RMS cells, as well as the ARMS specific fusion proteins PAX3/7-FOXO1. Our novel findings on TBX2 highlight the significant roles of TBX2 in muscle development and adult muscle regeneration, where TBX2 represses MRF activities to inhibit myogenic differentiation and promote proliferation of myoblasts. Also, our work establishes essential oncogene effects of TBX2 in driving and maintaining RMS proliferation and tumorigenesis by repressing cell cycle regulatory factors, p21 and p19/p14, and tumor suppressor of PTEN. Therefore, this work provides an exciting opportunity for development of new therapeutic treatments for TBX2 driven RMS cancer.

Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the molecular defects present in these cells that block myogenesis. We have found MEF2D is absent in RMS cell lines representing both major subtypes of RMS and primary cells derived from an embryonal RMS mice model. We have shown that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find MEF2D cannot bind to muscle specific gene promoters. Exogenous expression of MEF2D activates muscle specific luciferase constructs, upregulates p21 expression and increases muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility, anchorage independent growth in vitro, and tumor growth in vivo by xenograft assay. We also have found MEF2C is deregulated in rhabdomyosarcoma with the aberrant alternative splicing. We have shown that exon α in MEF2C is aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 being highly downregulated in RMS cells compared with normal myoblasts. We find that MEF2Cα1 is the ubiquitously expressed isoform which exhibits no myogenic activity and that MEF2Cα2, the muscle specific MEF2C isoform, is required for efficient differentiation. Compared with MEF2Cα2, MEF2Cα1 more strongly interacts with and recruits HDAC5 to myogenic gene promoters to repress muscle specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increases myogenic activity and promotes differentiation in RMS cells. We have also identified a serine protein kinase, SRPK3, which is downregulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage independent growth of RMS cells. The NAC complex performs many diverse biological functions, and the deregulation of its subunits has been correlated with many cancers. We sought to understand the function of the NAC complex in normal myogenesis and tumor progression in rhabdomyosarcoma cells. We found that the muscle specific subunit of the NAC complex, skNAC, which is the alternatively spliced isoform of NACα, was induced in normal cells and downregulated in RMS cells, while BTF3, also known as NACβ, was induced in normal cells and severely downregulated in RMS cells. We also showed that skNAC associated with muscle specific promoters together with BTF3 in differentiated normal cells, and this association was dependent on the expression of BTF3. We further investigated the involvement of skNAC in RMS progression. We found that the muscle specific expressed methyltransferase Smyd1 was nuclear localized in RMS cells and its interaction partner skNAC was switched with corepressors (HDAC1 and TBX2). We also confirmed the expression of skNAC was regulated by the splicing factor kinase SRPK3 and overexpression of SPRK3 induced skNAC expression and muscle differentiation in RMS cells. We also confirmed the overexpression of BTF3 in patient RMS tumors and depletion of BTF3 induced apoptosis in RMS cells and decrease RMS cell survival. BTF3 depletion also sensitized TRAIL induced cell apoptosis in RMS cells. However, BTF3 played a different role in normal cells. Deletion of BTF3 in C2C12 cells does not induce cell apoptosis, which suggests BTF3 functions as an anti-apoptosis factor in RMS cells and could be used as a cancer specific therapeutic target in RMS cells.

Deformed epidermal autoregulatory factor -1 (DEAF-1) is a transcription factor mapping to the chromosomal region 11p15.5, a region associated with loss of heterozygosity (LOH) in human cancers. Potential DEAF-1 binding motifs were identified in the PTEN promoter and the ability of DEAF-1 to regulate PTEN gene expression was investigated. DEAF-1 increased transcription 10-14 fold with PTEN sequences between -429 and -221, while mutations in the DNA binding domain (DEAF-ADWA) and nuclear localization signal of DEAF-1 abolished this increase. DEAF-1 was shown to bind sequences between -339 and -233. Rabdomyosarcoma (RD) stable cell lines with inducible expression of DEAF-1 and DEAF-1-ADWA were produced. Increased DEAF-1 expression had no significant effects on PTEN RNA expression or cell proliferation when compared to controls, but did increase susceptibility to UV-induced apoptosis. These studies suggest that DEAF-1 may contribute to the regulation of PTEN gene expression, but other factors may play a more significant role.

TBX2 and TBX3, which function as repressors, are members of the T-Box transcription factor family which are conserved throughout the metazoan lineage. TBX2 is highly expressed in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, and many other cancers. Previously, our lab dissected the oncogenic properties of TBX2 and its regulation of p14, p21 and PTEN. TBX3 is also expressed in some cancer types, however, its expression profile in RMS is severely down-regulated. TBX3 is shown to repress TBX2 in chondrocytes, but the characterization and regulation of TBX3 is poorly understood in the muscle lineage. Polycomb Repressive Complex 2 (PRC2), a gene silencing complex, acts to methylate histone H3 lysine 27 (H3K27me) of target gene promoters. The catalytic subunit of PRC2, EZH2, is up-regulated in RMS and data from our lab has shown that depletion of EZH2 up-regulated TBX3 and down-regulated TBX2 in C2C12 cells, an immortalized murine cell line. The hypothesis of this project was that there would be a PRC2-TBX3-TBX2 axis in RMS cells. To examine if TBX3 represses TBX2, TBX3 was transiently expressed in RMS cells representing both subtypes of RMS and we found that TBX2 was downregulated in each cell line. In a stable RH30 cell line with ectopic TBX3, TBX2 was down-regulated and PTEN expression was up-regulated. To determine if TBX2 repression by TBX3 was direct, a TBX3 ChIP assay was performed on the TBX2 promoter as well as the PTEN promoter. We found a strong enrichment of TBX3 on the TBX2 promoter but not on the PTEN promoter. Accordingly, we also observed that TBX3 over-expression impaired tumorigenesis through reduced cell proliferation, migration, and anchorage dependent growth. Also, we found that a stable RD cell line with ectopic TBX3 could promote differentiation, strongly suggesting that these results could have therapeutic value. Next, a shEZH2 plasmid was transfected into RMS cell lines ask if TBX3 was regulated by the PRC2 complex as we had observed in C2C12 cells. Just as we hypothesized, TBX3 was up-regulated and TBX2 was down-regulated. Similar to the previous TBX3 overexpression experiments, the EZH2 depleted RMS cell lines also showed decreased cell proliferation and migration rate. Also, an EZH2 knock down treatment induced differentiation in RMS cell lines. Therefore, understanding this potent regulation axis could provide an excellent opportunity for treatment of RMS cancer in the future.

Cancer immunotherapies focused on tumor-specific T cell responses are promising alternatives to chemotherapy/radiotherapy for various cancers as they can be engineered to specifically target tumours and establish long-term surveillance against relapsing tumours. The premise for the application of active immunotherapies is the recognition of tumor-specific and/or tumor-associated antigens by the immune system. Approaches that have been explored to this end include cancer vaccines and gene therapy/autologous cell transfer (T-cell receptor (TCR) or chimeric antigen receptor (CAR)-based). This study evaluated the use of oncolytic rhabdovirus-based vaccines (ORV) for the treatment of sarcoma with a focus on rhabdomyosarcoma (RMS). Sarcomas are amenable to to oncolytic virus (OV) infection and generate robust T-cell responses against tumour antigens. The ORV strategy undergoing clinical evaluation uses a prime-boost vaccine whereby a non-replicating adenovirus serotype 5 vector (Ad5) encoding an antigen is administered as a priming agent and boosted with a rhabdovirus encoding the same antigen (NCT02285816). However, the prevalence of pre-existing immunity to Ad5 in patients serves as an exclusion criterion and limits its effectiveness as a priming agent. To this end, we have shown that an alternative priming agent and antigen delivery vehicle, anti-DEC205 (aDEC205), targets antigens directly to dendritic cells (DCs), inducing robust immune responses. However, a lack of targetable antigens and methods to identify antigens is a limiting step for the application of ORVs for sarcoma. Thus, the identification of immunogenic sarcoma antigens is a critical step for the study ORVs. Current methodologies have important drawbacks in that they can be prohibitively time-consuming, complex or are ineffective in coupling antigen discovery and immunogenicity. Presented herein is the study of a novel methodology for the discovery of immunogenic antigens by probing for T cell activation marker CD107a and isolation by flow cytometry. In parallel, RMS antigen discovery was also performed via peptide elution and mass spectrometry resulting in the identification of 24 novel murine RMS antigens. Ultimately, therapeutic vaccination with a subset of these antigens encoded into DEC205 and ORV did not yield immune responses in a pre-clinical model; however, this research established immunization tools for further study of immunotherapy in RMS.

No description available.

The Hippo pathway is a master regulator of cell proliferation and organ size, namely through regulation of transcriptional co-activators YAP and TAZ which bind TEAD1-4 transcription factors. The Hippo effector YAP is dysregulated in many human solid tumours including rhabdomyosarcoma and oesophageal cancer. Additionally, persistent hyperactivity of YAP in activated but not quiescent satellite cells can give rise to embryonal rhabdomyosarcoma. However, the question of exactly how YAP acts as an oncogene and actively gives rise to tumour progression in these cancers remains unknown. In this thesis I characterised the mechanisms which determine the functional role of YAP in driving instability in the genome. Secondly, lentiviral mediated knockdown of YAP is performed to determine and investigate its effect on tumorigenesis. Thirdly, gene sets from constitutive YAP S127A induced mouse ERMS tumours subjected to array-CGH were further analysed. Finally, I cloned chicken Yap1, Tead1 and Fstl5 to identify its role during chick embryonic development, by the retroviral mediated loss of function approach. The results demonstrated that constitutive YAP S127A expression in-vitro as well as in-vivo induces chromosomal instability by increasing the rate of mitotic chromosome segregation errors and copy number alterations of oncogenes and other cancer related genes. Recurrent copy number gains of the p53 inhibitor Mdm2 were observed in YAP S127A-driven ERMS tumours. Moreover, lentiviral mediated YAP knockdown showed significant reduction in proliferation, migration and invasion as well as transformation potential in human cultured cancer cells. Moreover, retroviral YAP S127A expression during early stages of chick embryo development did not lead to an overt phenotype and showed poor survival. Additionally, I have cloned RCAS-RNAi vectors to study the loss of function effect on Hippo targets and Fstl5 during chicken embryo development. Collectively, my data provides insight into the mechanisms with which YAP could drive tumorigenesis and that YAP knockdown can be considered a potential therapeutic target to reduce cancer progression.

INTRODUÇÃO: Rabdomiossarcoma (RMS) representa o sarcoma de partes moles mais freqüente da infância, havendo poucas informações a seu respeito em países em desenvolvimento. OBJETIVOS: Estudar o perfil demográfico, social, clínico, biológico e patológico de pacientes portadores de rabdomiossarcoma tratados em uma instituição brasileira. Estimar a probabilidade acumulada de sobrevida global (SG) e de sobrevida livre de eventos (SLE) em 60 meses, assim como identificar fatores prognósticos. CASUÍSTICA E MÉTODOS: Foram analisados retrospectivamente 163 pacientes, no período de 1986 a 2004, que receberam tratamento multimodal seguindo orientações do protocolo Intergrupo para RMS III e IV. O estudo do percentual de positividade nuclear da miogenina foi realizado em 85 casos. RESULTADOS: As localizações da doença mais freqüentes foram cabeça e pescoço em 73 (44,7%), extremidade em 28 (17,2%), trato geniturinário em 25 (15,3%) e retroperitônio em 14 (8,6%) pacientes. O subtipo histológico foi embrionário em 99 (60,75%) pacientes e alveolar/sarcoma indiferenciado em 65 (38,7%). Foi observada positividade nuclear da imunoistoquímica com miogenina (> 75% das células) em 80% dos casos com RMS alveolar. O tamanho tumoral foi > 5 cm em 89%, com invasividade (T2) em 80,4% e comprometimento de linfonodos regionais em 45 (27,6%) pacientes. A maioria dos pacientes encontrava-se nos grupos clínicos III (49,1%) e IV (39,3%). O estado nutricional foi avaliado com o percentil do índice de massa corporal (IMC), sendo encontrado baixo peso (< 10º percentil) em 49 (30%) pacientes. Com tempo mediano de seguimento de 32 meses, as probabilidades acumuladas de SG e SLE para o grupo todo foram 48,6% e 42,2%, respectivamente. Nos pacientes com GC I + II a SLE foi de 73,3%; naqueles com GC III foi 57,5% e nos com GC IV foi 14,9% (p = 0,001). Na análise univariada, a SG em 60 meses foi influenciada negativamente por idade < 1 ano e maior ou igual a 10 anos, cor da pele preta, doença metastática (GC IV), um ou dois e mais sítios de metástases, local do tumor primário em extremidade, linfonodo N1 ou NX, invasividade e subtipo histológico alveolar. Na análise multivariada, foram fatores prognósticos Resumo independentes para SG: idade < 1 ano (p = 0,022) e maior ou igual a 10 anos (p = 0,069), um ou dois e mais sítios de metástase (p < 0,001) e percentil do IMC menor ou igual a 10 (p = 0,027); para SLE, IMC menor ou igaul a 10º percentil (p = 0,036) e presença de linfondo regional N1 e NX (p < 0,001); para SLE nos pacientes sem metástases, IMC menor ou igual a 10º percentil (p = 0,003) e presença de linfondo regional N1 (p = 0,003) e NX (p = 0,002). O tamanho tumoral, apesar de não ter entrado no modelo multivariado, foi um dos fatores prognósticos mais importantes. O estudo da positividade da miogenina não teve significado prognóstico. CONCLUSÕES: Esses resultados sugerem que além das características da doença, fatores nutricionais e socioeconômicos devem ser considerados no planejamento do tratamento e na análise de fatores prognósticos. Doença metastática está associada a prognóstico reservado. Investir no diagnóstico precoce é fundamental. / INTRODUCTION: Rhabdomyosarcoma (RMS) represents the most frequent soft tissue tumor in childhood. There are few reports on this disease in developing countries. PURPOSE: To analyze demographic, socio-economic, clinical, biological and pathological variables at a Brazilian institution. To estimate overall survival (OS) and event-free survival (EFS) in 60 months and identify prognostic factors. CASUISTICS AND METHODS: One hundred and sixty-three patients with rhabdomyosarcoma who had received multimodal treatment according to Intergroup protocols for rhabdomyosarcoma - IRS III and IV -were retrospectively analyzed from 1986 to 2004. The nutritional status was evaluated with the body mass index (BMI) percentile and less than 10 was considered low weight. Immunohistochemistry with myogenin was performed in 85 patients for the percentage of nuclear positivity. RESULTS: The frequent primary sites in patients were head and neck in 73 (44.7%), extremity in 28 (17.2%), genitourinary tract in 25 (15.3%) and retroperitoneum in 14 (8.6%). The histological subtype was embryonal in 99 (60.75%) and alveolar in 65 (38.7%) patients. Nuclear positivity for myogenin (> 75%), was observed in 80% of alveolar RMS patients. Tumor size > 5 cm was seen in 89%, invasivity (T2) in 80.4% and regional lymph node involvement in 45 (27.6%) patients. Most patients were in clinical groups (CG) III (49.1%) and IV (39.3%). The BMI was below the 10th percentile in 49 (30%) patients. With a median follow-up of 32 months, estimated OS and EFS for the whole group were 48.6% and 42.2%, respectively. The EFS for patients in CG I + II, III and IV was 73.3%, 57.5% and 14.9%, respectively (p = 0.001). Univariate analysis showed that OS in 60 months was adversely influenced by age < 1 year and maior ou igual a 10 year, black skin, metastatic disease, one or two and more metastatic sites, extremity as primary site, lymph node N1 or NX, invasivity, and histological subtype alveolar. Multivariate analysis showed that independent prognostic factors for OS were age < 1 year (p = 0.022) and maior ou igual a 10 years (p = 0.069), one or two and more metastatic sites (p < 0.001), BMI percentile menor ou igual 10 (p = 0.027); independent prognostic factors for EFS were BMI < 10th percentile (p = 0.036) and the presence of regional lymph node N1 e NX (p < 0.001); for EFS in Summary non-metastatic patients, BMI < 10th percentile (p = 0.003) and the presence of regional lymph nodes N1 (p = 0.003) and NX (p = 0.002). Tumor size was one of the most important factors, although not selected for the multivariate model. The positivity of myogenin had no prognostic significance. CONCLUSIONS: The results of this study suggest that besides disease characteristics, nutritional and socio-economic factors should be considered in planning treatment and in the analysis of prognostic factors. Metastatic disease is associated with bad prognosis. All efforts should be taken to provide early diagnosis.

INTRODUÇÃO: Rabdomiossarcoma (RMS) representa o sarcoma de partes moles mais freqüente da infância, havendo poucas informações a seu respeito em países em desenvolvimento. OBJETIVOS: Estudar o perfil demográfico, social, clínico, biológico e patológico de pacientes portadores de rabdomiossarcoma tratados em uma instituição brasileira. Estimar a probabilidade acumulada de sobrevida global (SG) e de sobrevida livre de eventos (SLE) em 60 meses, assim como identificar fatores prognósticos. CASUÍSTICA E MÉTODOS: Foram analisados retrospectivamente 163 pacientes, no período de 1986 a 2004, que receberam tratamento multimodal seguindo orientações do protocolo Intergrupo para RMS III e IV. O estudo do percentual de positividade nuclear da miogenina foi realizado em 85 casos. RESULTADOS: As localizações da doença mais freqüentes foram cabeça e pescoço em 73 (44,7%), extremidade em 28 (17,2%), trato geniturinário em 25 (15,3%) e retroperitônio em 14 (8,6%) pacientes. O subtipo histológico foi embrionário em 99 (60,75%) pacientes e alveolar/sarcoma indiferenciado em 65 (38,7%). Foi observada positividade nuclear da imunoistoquímica com miogenina (> 75% das células) em 80% dos casos com RMS alveolar. O tamanho tumoral foi > 5 cm em 89%, com invasividade (T2) em 80,4% e comprometimento de linfonodos regionais em 45 (27,6%) pacientes. A maioria dos pacientes encontrava-se nos grupos clínicos III (49,1%) e IV (39,3%). O estado nutricional foi avaliado com o percentil do índice de massa corporal (IMC), sendo encontrado baixo peso (< 10º percentil) em 49 (30%) pacientes. Com tempo mediano de seguimento de 32 meses, as probabilidades acumuladas de SG e SLE para o grupo todo foram 48,6% e 42,2%, respectivamente. Nos pacientes com GC I + II a SLE foi de 73,3%; naqueles com GC III foi 57,5% e nos com GC IV foi 14,9% (p = 0,001). Na análise univariada, a SG em 60 meses foi influenciada negativamente por idade < 1 ano e maior ou igual a 10 anos, cor da pele preta, doença metastática (GC IV), um ou dois e mais sítios de metástases, local do tumor primário em extremidade, linfonodo N1 ou NX, invasividade e subtipo histológico alveolar. Na análise multivariada, foram fatores prognósticos Resumo independentes para SG: idade < 1 ano (p = 0,022) e maior ou igual a 10 anos (p = 0,069), um ou dois e mais sítios de metástase (p < 0,001) e percentil do IMC menor ou igual a 10 (p = 0,027); para SLE, IMC menor ou igaul a 10º percentil (p = 0,036) e presença de linfondo regional N1 e NX (p < 0,001); para SLE nos pacientes sem metástases, IMC menor ou igual a 10º percentil (p = 0,003) e presença de linfondo regional N1 (p = 0,003) e NX (p = 0,002). O tamanho tumoral, apesar de não ter entrado no modelo multivariado, foi um dos fatores prognósticos mais importantes. O estudo da positividade da miogenina não teve significado prognóstico. CONCLUSÕES: Esses resultados sugerem que além das características da doença, fatores nutricionais e socioeconômicos devem ser considerados no planejamento do tratamento e na análise de fatores prognósticos. Doença metastática está associada a prognóstico reservado. Investir no diagnóstico precoce é fundamental. / INTRODUCTION: Rhabdomyosarcoma (RMS) represents the most frequent soft tissue tumor in childhood. There are few reports on this disease in developing countries. PURPOSE: To analyze demographic, socio-economic, clinical, biological and pathological variables at a Brazilian institution. To estimate overall survival (OS) and event-free survival (EFS) in 60 months and identify prognostic factors. CASUISTICS AND METHODS: One hundred and sixty-three patients with rhabdomyosarcoma who had received multimodal treatment according to Intergroup protocols for rhabdomyosarcoma - IRS III and IV -were retrospectively analyzed from 1986 to 2004. The nutritional status was evaluated with the body mass index (BMI) percentile and less than 10 was considered low weight. Immunohistochemistry with myogenin was performed in 85 patients for the percentage of nuclear positivity. RESULTS: The frequent primary sites in patients were head and neck in 73 (44.7%), extremity in 28 (17.2%), genitourinary tract in 25 (15.3%) and retroperitoneum in 14 (8.6%). The histological subtype was embryonal in 99 (60.75%) and alveolar in 65 (38.7%) patients. Nuclear positivity for myogenin (> 75%), was observed in 80% of alveolar RMS patients. Tumor size > 5 cm was seen in 89%, invasivity (T2) in 80.4% and regional lymph node involvement in 45 (27.6%) patients. Most patients were in clinical groups (CG) III (49.1%) and IV (39.3%). The BMI was below the 10th percentile in 49 (30%) patients. With a median follow-up of 32 months, estimated OS and EFS for the whole group were 48.6% and 42.2%, respectively. The EFS for patients in CG I + II, III and IV was 73.3%, 57.5% and 14.9%, respectively (p = 0.001). Univariate analysis showed that OS in 60 months was adversely influenced by age < 1 year and maior ou igual a 10 year, black skin, metastatic disease, one or two and more metastatic sites, extremity as primary site, lymph node N1 or NX, invasivity, and histological subtype alveolar. Multivariate analysis showed that independent prognostic factors for OS were age < 1 year (p = 0.022) and maior ou igual a 10 years (p = 0.069), one or two and more metastatic sites (p < 0.001), BMI percentile menor ou igual 10 (p = 0.027); independent prognostic factors for EFS were BMI < 10th percentile (p = 0.036) and the presence of regional lymph node N1 e NX (p < 0.001); for EFS in Summary non-metastatic patients, BMI < 10th percentile (p = 0.003) and the presence of regional lymph nodes N1 (p = 0.003) and NX (p = 0.002). Tumor size was one of the most important factors, although not selected for the multivariate model. The positivity of myogenin had no prognostic significance. CONCLUSIONS: The results of this study suggest that besides disease characteristics, nutritional and socio-economic factors should be considered in planning treatment and in the analysis of prognostic factors. Metastatic disease is associated with bad prognosis. All efforts should be taken to provide early diagnosis.

<p>Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma and demonstrates features of skeletal muscle. Of the two predominant (pediatric) subtypes, embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a 5-year survival rate of <50%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, we aimed to identify proteins that are downstream from or cooperate with PAX3-FOXO1 (PF) to enable tumorigenesis with the hope that these proteins may be more amenable to pharmacological inhibition.</p><p>First, in a microarray analysis of the transcriptomes of human skeletal muscle myoblasts expressing PF, we observed alterations of several Wnt pathway genes, including the Wnt inhibitor Secreted Frizzled Related Protein 3 (SFRP3). Loss-of-function studies interrogated the role of SFRP3 in human aRMS cell lines using shRNAs. Suppression of SFRP3 inhibited aRMS cell growth, reduced proliferation accompanied by a G1 arrest and induction of p21, and induced apoptosis. SFRP3 suppression modestly increased Wnt signaling; however, activation of the Wnt pathway in human aRMS cells in vitro and in a xenograft murine model of aRMS in vivo only partially recapitulated the phenotype observed with SFRP3 suppression. To identify other signaling pathways downstream of SFRP3 signaling, we conducted an oncogenic signaling pathways screen and a microarray. In the former, we identified Notch signaling as conferring resistance to SFRP3 suppression-mediated decreased cell growth and confirmed Notch crosstalk with Wnt signaling and SFRP3 in aRMS cells. In the latter, SFRP3 suppression increased genes associated with skeletal muscle differentiation and decreased those associated with cell cycle progression. </p><p>Second, we established a role for SFRP3 in a conditional xenograft murine model of aRMS. Doxycycline-inducible suppression of SFRP3 reduced aRMS tumor growth and weight by more than three-fold. Analysis of the tumors by qPCR and IHC revealed an increase in myogenic differentiation and β-catenin signaling. The combination of SFRP3 suppression and vincristine was more effective at reducing aRMS cell growth in vitro than either treatment alone, and ablated tumorigenesis in vivo. In conclusion, SFRP3 is necessary for the growth of human aRMS cells both in vitro and in vivo and is a promising new target for investigation in aRMS.</p> / Dissertation