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BACKGROUND: Cancer stem/Initiating cell (CS/IC) hypothesis argues that CS/ICs are responsible of tumour initiation, drug resistance, metastasis or disease relapse. Their detection in several cancers supports this concept. However, their origin is still misunderstood. Cell fusion is shown to take part in the formation of CS/ICs, i.e. fusion between mesenchymal stem cell and cancer cell. In a previous paper, we described that fusion leads to hybrids with metastatic capacity. This process triggered genomic rearrangements in hybrid cells together with increased metastasis development. Here, we hypothesize that cell fusion could be strong enough to provoke a cellular reprogramming and the acquisition of CS/IC properties, promoting metastasis formation. METHODS: After spontaneous cell fusion between E6E7 (IMR90 with the oncogenes E6 and E7) and RST (IMR90 fully transformed) cell lines, hybrid cells were selected by dual antibiotic selection. Cancer stem cells capacities were evaluated regarding capacity to form spheres, expression of stem cell markers and the presence of ALDHhigh cells. RESULTS: Our data show that after cell fusion, all hybrids contain a percentage of cells with CS/ICs properties, regarding. Importantly, we lastly showed that NANOG inhibition in H1 hybrid decreases this migration capacity while having no effect on the corresponding parental cells. CONCLUSIONS: Altogether these results indicate that the combination of CS/ICs properties and genomic rearrangement in hybrids is likely to be key to tumour progression.
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Carcinogênese/patologia , Reprogramação Celular , Células-Tronco Mesenquimais/patologia , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Fusão Celular , Linhagem Celular Tumoral , Humanos , Células Híbridas , Esferoides CelularesRESUMO
BACKGROUND: Cell-to-cell fusion is emerging as a key element of the metastatic process in various cancer types. We recently showed that hybrids made from the spontaneous merging of pre-malignant (IMR90 E6E7, i.e. E6E7) and malignant (IMR90 E6E7 RST, i.e. RST) mesenchymal cells recapitulate the main features of human undifferentiated pleomorphic sarcoma (UPS), with a highly rearranged genome and increased spreading capacities. To better characterize the intrinsic properties of these hybrids, we investigated here their metabolic energy profile compared to their parents. RESULTS: Our results unveiled that hybrids harbored a Warburg-like metabolism, like their RST counterparts. However, hybrids displayed a much greater metabolic activity, enhancing glycolysis to proliferate. Interestingly, modifying the metabolic environmental conditions through the use of 5-aminoimidazole-4-carbox-amide-1-ß-D-ribofuranoside (AICAR), an activator of the 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), specifically reduced the growth of hybrids, and also abrogated the invasive capacity of hybrids displaying enhanced glycolysis. Furthermore, AICAR efficiently blocked the tumoral features related to the aggressiveness of human UPS cell lines. CONCLUSION: Altogether, our findings strongly suggest that hybrids rely on higher energy flux to proliferate and that a drug altering this metabolic equilibrium could impair their survival and be potentially considered as a novel therapeutic strategy.
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Metabolismo Energético , Células Gigantes/metabolismo , Células Gigantes/patologia , Células Híbridas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Invasividade Neoplásica , Neoplasias/genética , Processos NeoplásicosRESUMO
BACKGROUND: The aim of this study was to explore the efficacy and define mechanisms of action of PRIMA-1(MET) as a TP53 targeted therapy in soft-tissue sarcoma (STS) cells. METHODS: We investigated effects of PRIMA-1(MET) on apoptosis, cell cycle, and induction of oxidative stress and autophagy in a panel of 6 STS cell lines with different TP53 status. RESULTS: Cell viability reduction by PRIMA-1(MET) was significantly observed in 5 out of 6 STS cell lines. We found that PRIMA-1(MET) was capable to induce cell death not only in STS cells harboring mutated TP53 but also in TP53-null STS cells demonstrating that PRIMA-1(MET) can induce cell death independently of TP53 in STS cells. We identified an important role of reactive oxygen species (ROS), involved in PRIMA-1(MET) toxicity in STS cells leading to a caspase-independent cell death. ROS toxicity was associated with autophagy induction or JNK pathway activation which represented potential mechanisms of cell death induced by PRIMA-1(MET) in STS. CONCLUSIONS: PRIMA-1(MET) anti-tumor activity in STS partly results from off-target effects involving ROS toxicity and do not deserve further development as a TP53-targeted therapy in this setting.
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Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Quinuclidinas/farmacologia , Sarcoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sarcoma/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: The biologic heterogeneity of soft tissue sarcomas (STS), even within histological subtypes, complicates treatment. In earlier studies, gene expression patterns that distinguish two subsets of clear cell renal carcinoma (RCC), serous ovarian carcinoma (OVCA), and aggressive fibromatosis (AF) were used to separate 73 STS into two or four groups with different probabilities of developing metastatic disease (PrMet). This study was designed to confirm our earlier observations in a larger independent data set. METHODS: We utilized these gene sets, hierarchical clustering (HC), and Kaplan-Meier analysis, to examine 309 STS, using Affymetrix chip expression profiling. RESULTS: HC using the combined AF-, RCC-, and OVCA-gene sets identified subsets of the STS samples. Analysis revealed differences in PrMet between the clusters defined by the first branch point of the clustering dendrogram (p = 0.048), and also among the four different clusters defined by the second branch points (p < 0.0001). Analysis also revealed differences in PrMet between the leiomyosarcomas (LMS), dedifferentiated liposarcomas (LipoD), and undifferentiated pleomorphic sarcomas (UPS) (p = 0.0004). HC of both the LipoD and UPS sample sets divided the samples into two groups with different PrMet (p = 0.0128, and 0.0002, respectively). HC of the UPS samples also showed four groups with different PrMet (p = 0.0007). HC found no subgroups of the LMS samples. CONCLUSIONS: These data confirm our earlier studies, and suggest that this approach may allow the identification of more than two subsets of STS, each with distinct clinical behavior, and may be useful to stratify STS in clinical trials and in patient management.
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Expressão Gênica , Heterogeneidade Genética , Metástase Neoplásica/genética , Sarcoma/patologia , Humanos , Probabilidade , Sarcoma/classificação , Sarcoma/genética , Sitios de Sequências RotuladasRESUMO
INTRODUCTION: Liposomal irinotecan promotes controlled sustained release of irinotecan (CPT-11), therefore, we hypothesize that the therapeutic index (quantitative measurement of the relative efficacy/safety ratio of a drug) will be higher for liposomal than non-liposomal irinotecan. METHODS: We compared the therapeutic indexes of liposomal and non-liposomal irinotecan in mice bearing subcutaneous patient-derived xenograft (PDX) pancreatic tumors under dosing regimens approximating the clinical setting. Following preliminary drug sensitivity/antitumor activity analyses on three PDX tumor models, one model was selected for analyses of efficacy, biomarker, toxicology, pharmacokinetics in mice receiving liposomal irinotecan (2.5, 10, 50 mg/kg/week) or non-liposomal irinotecan (10, 25, 50 mg/kg/week). The maximum tolerated dose (MTD) for each treatment was 50 mg/kg/week. RESULTS: Using the selected IM-PAN-001 model at the MTD (both treatments, 50 mg/kg/week), antitumor activity, phospho-histone gamma-H2AX protein staining in cancer cell nuclei, histological tumor regression, and plasma levels of CPT-11 and its active metabolite SN-38 after 24 h were greater with liposomal than non-liposomal irinotecan, but tumor SN-38 levels were similar. At the lowest doses assessed, antitumor activity, histological tumor regression, and jejunum and bone marrow toxicity were similar. Based on these findings, liposomal and non-liposomal irinotecan had therapeutic indexes of 20 and 5, respectively. CONCLUSION: This non-clinical study showed a fourfold broader therapeutic index with liposomal than non-liposomal irinotecan in mice bearing IM-PAN-001 PDX pancreatic tumors, even at optimal dosing for the two drugs. These findings support the clinical benefit observed with liposomal irinotecan in patients with pancreatic cancer.
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BACKGROUND: The management of soft-tissue sarcoma (STS) relies on a multidisciplinary approach involving specialized oncological surgery combined with other adjuvant therapies to achieve optimal local disease control. Purpose and Results: Genomic and transcriptomic pseudocapsules of 20 prospective sarcomas were analyzed and revealed to be correlated with a higher risk of recurrence after surgery. CONCLUSIONS: A peritumoral environment that has been remodeled and infiltrated by M2 macrophages, and is less expressive of healthy tissue, would pose a significant risk of relapse and require more aggressive treatment strategies.
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Over the past decade, comprehensive genomic studies demonstrated that leiomyosarcomas and most of the tumors previously labeled as 'malignant fibrous histiocytomas' share complex karyotypes and genomic profiles, and can be referred to as 'sarcomas with complex genomics'. We recently reported a series of 160 sarcomas with complex genomics such as leiomyosarcomas, myxofibrosarcomas, pleomorphic liposarcomas/rhabdomyosarcomas and undifferentiated pleomorphic sarcomas. These tumors present with a frequent loss of chromosome 10 region encompassing the tumor suppressor gene PTEN. In the present study, we assessed PTEN genomic level and protein expression in this large series of sarcomas with complex genomics, as well as activation of downstream pathways. PTEN partial genomic loss was observed in only 46% of tumors, especially in well-differentiated leiomyosarcomas, whereas up to 68% of these tumors demonstrate a loss of protein expression on western blot analysis. Specific discrepancies in PTEN immunohistochemical results suggested bias in this latter technique. PTEN mutations were rare, with only 4 point mutations in the 65 samples studied. Subsequent activation of AKT and mTOR pathways was only observed in 2 out of 3 of PTEN-deleted tumors. On the other hand, RICTOR, a major component of the mTOR complex 2, was significantly overexpressed in well-differentiated leiomyosarcomas. These results, confirmed on tissue micro-array immunohistochemical analysis of 459 sarcomas, could suggest a link between RICTOR overexpression and leiomyosarcomas oncogenesis. As therapeutics directed against the mTOR pathway are assessed in sarcomas, RICTOR overexpression in sarcomas and its links to therapeutic response need to be assessed.
Assuntos
Proteínas de Transporte/genética , Diferenciação Celular , Leiomiossarcoma/genética , Lipossarcoma/genética , PTEN Fosfo-Hidrolase/genética , Tumor de Músculo Liso/genética , Serina-Treonina Quinases TOR/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Hibridização Genômica Comparativa , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Leiomiossarcoma/classificação , Leiomiossarcoma/metabolismo , Lipossarcoma/classificação , Lipossarcoma/patologia , Masculino , Pessoa de Meia-Idade , Músculo Liso/metabolismo , Músculo Liso/patologia , PTEN Fosfo-Hidrolase/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Tumor de Músculo Liso/classificação , Tumor de Músculo Liso/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Análise Serial de TecidosRESUMO
Adult soft tissue sarcomas (STS) are rare tumours of mesenchymal lineage. Based on cytogenetic and comparative genomic hybridization (CGH) data, they can be divided into 'STS with simple genomics', displaying a characteristic genetic alteration, and 'STS with complex genomics' (SCG), where multiple genomic alterations occur. This latter group is mostly composed of leiomyosarcomas (LMS) and pleiomorphic undifferentiated tumours previously labelled as 'malignant fibrous histiocytomas' (MFH), corresponding in fact to myxofibrosarcomas (MFS), pleiomorphic liposarcomas/rhabdomyosarcomas (P-LPS, P-RMS), and undifferentiated pleiomorphic sarcomas (UPS). Their pathobiology is still not well understood, leading to challenges in diagnosis and therapeutic management. We report here a comprehensive study encompassing array-CGH and transcriptome analysis data of a large series of 160 SCG. Non-supervised clustering of transcriptome data led to the identification of five groups of tumours, one of them (group A) corresponding to well-differentiated LMS and the other four (B-E) to 'MFH' and poorly differentiated LMS. Welch analysis of transcriptome data in these groups allowed us to retrieve several genes of potential interest. Among them, RB1 alteration is a constant thread in SCG, often associated with RBL2 loss. PTEN tumour suppressor deletion would also stand out as a major recurrent event, especially in groups A, C, and D. The WNT canonical pathway could be potentially involved, as demonstrated by up-regulation of one of its inhibitors, DKK1, in groups D and E, whereas DKK1 is significantly down-regulated in groups A, B, and C. These data suggest a very complex interplay between pathways downstream of PTEN and the WNT canonical pathway, providing new hints about SCG pathobiology and their potential therapeutic targets.
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Sarcoma/genética , Neoplasias de Tecidos Moles/genética , Adulto , Idoso , Transformação Celular Neoplásica/genética , Análise por Conglomerados , Hibridização Genômica Comparativa/métodos , Feminino , Deleção de Genes , Perfilação da Expressão Gênica/métodos , Genômica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Leiomiossarcoma/genética , Leiomiossarcoma/patologia , Masculino , Pessoa de Meia-Idade , PTEN Fosfo-Hidrolase/genética , Proteína do Retinoblastoma/genética , Sarcoma/patologia , Neoplasias de Tecidos Moles/patologiaRESUMO
Alterations of the p53 pathway are among the most frequent aberrations observed in human cancers. We have performed an exhaustive analysis of TP53, p14, p15, and p16 status in a large series of 143 soft tissue sarcomas, rare tumors accounting for around 1% of all adult cancers, with complex genetics. For this purpose, we performed genomic studies, combining sequencing, copy number assessment, and expression analyses. TP53 mutations and deletions are more frequent in leiomyosarcomas than in undifferentiated pleomorphic sarcomas. Moreover, 50% of leiomyosarcomas present TP53 biallelic inactivation, whereas most undifferentiated pleomorphic sarcomas retain one wild-type TP53 allele (87.2%). The spectrum of mutations between these two groups of sarcomas is different, particularly with a higher rate of complex mutations in undifferentiated pleomorphic sarcomas. Most tumors without TP53 alteration exhibit a deletion of p14 and/or lack of mRNA expression, suggesting that p14 loss could be an alternative genotype for direct TP53 inactivation. Nevertheless, the fact that even in tumors altered for TP53, we could not detect p14 protein suggests that other p14 functions, independent of p53, could be implicated in sarcoma oncogenesis. In addition, both p15 and p16 are frequently codeleted or transcriptionally co-inhibited with p14, essentially in tumors with two wild-type TP53 alleles. Conversely, in TP53-altered tumors, p15 and p16 are well expressed, a feature not incompatible with an oncogenic process.
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Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Mutação/genética , Sarcoma/genética , Transdução de Sinais , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Adulto , Western Blotting , Hibridização Genômica Comparativa , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metilação de DNA , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
In a series of 404 adult soft tissue sarcomas, analyzed by array-CGH, we have observed in approximately 10% of them a genomic amplification of either chromosome bands 11q22 or 3p12. These two amplicons likely target the YAP1 and VGLL3 genes, respectively. Both genes encode proteins that are cofactors of the TEAD family of transcription factors. Very good correlations between amplification and expression levels were observed. Welch test analyses of transcriptome data demonstrate that tumors with amplicons share a large set of upregulated and downregulated genes. Inhibition of YAP1 and VGLL3 in cell lines with these amplifications/overexpressions leads to similar phenotypes: decrease of proliferation rate, and to a lesser extent decrease of migration properties. These data, and the fact that these amplicons are observed either in de-differentiated liposarcomas or in undifferentiated pleomorphic sarcomas, suggest that these genetics events could be involved in oncogenesis and progression of soft tissue sarcomas.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Amplificação de Genes , Fosfoproteínas/genética , Sarcoma/genética , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína 3 com Repetições IAP de Baculovírus , Linhagem Celular Tumoral , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 3 , Hibridização Genômica Comparativa , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Proteínas Inibidoras de Apoptose/genética , Lipossarcoma/diagnóstico , Lipossarcoma/genética , Lipossarcoma/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Sarcoma/diagnóstico , Sarcoma/patologia , Ubiquitina-Proteína Ligases , Proteínas de Sinalização YAPRESUMO
Desmoid tumors are fibroblastic/myofibroblastic proliferations. Previous studies reported that CTNNB1 mutations were detected in 84% and that mutations of the APC gene were found in several cases of sporadic desmoid tumors lacking CTNNB1 mutations. Forty tumors were analyzed by comparative genomic hybridization (CGH). Karyotype and fluorescence in situ hybridization revealed a nonrandom occurrence of trisomy 8 associated with an increased risk of recurrence. We report the first molecular characterization including a large series of patients. We performed array CGH on frozen samples of 194 tumors, and we screened for APC mutations in patients without CNNTB1 mutation. A high frequency of genomically normal tumors was observed. Four relevant and recurrent alterations (loss of 6q, loss of 5q, gain of 20q, and gain of Chromosome 8) were found in 40 out of 46 tumors with chromosomal changes. Gain of Chromosomes 8 and 20 was not associated with an increased risk of recurrence. Cases with loss of 5q had a minimal common region in 5q22.5 including the APC locus. Alterations of APC, including loss of the entire locus, and CTNNB1 mutation could explain the tumorigenesis in 89% of sporadic desmoids tumors and desmoids tumors occurring in the context of Gardner's syndrome. A better understanding of the pathogenetic pathways in the initiation and progression of desmoid tumors requires studies of 8q and 20q gains, as well as of 6q and 5q losses, and study of the Wnt/beta-catenin pathway.
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Neoplasias Abdominais/genética , Fibromatose Agressiva/genética , Neoplasias Abdominais/diagnóstico , Neoplasias Abdominais/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Distribuição de Qui-Quadrado , Criança , Pré-Escolar , Hibridização Genômica Comparativa/métodos , Feminino , Fibromatose Agressiva/diagnóstico , Fibromatose Agressiva/patologia , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Estimativa de Kaplan-Meier , Cariotipagem , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase , Gravidez , Análise de Sequência de DNA/métodos , beta Catenina/genéticaRESUMO
Cell fusion in tumor progression mostly refers to the merging of a cancer cell with a cell that has migration and immune escape capabilities such as macrophages. Here we show that spontaneous hybrids made from the fusion of transformed mesenchymal cells with partners from the same lineage undergo nonrecurrent large-scale genomic rearrangements, leading to the creation of highly aneuploid cells with novel phenotypic traits, including metastatic spreading capabilities. Moreover, in contrast to their parents, hybrids were the only cells able to recapitulate in vivo all features of human pleomorphic sarcomas, a rare and genetically complex mesenchymal tumor. Hybrid tumors not only displayed specific mesenchymal markers, but also combined a complex genetic profile with a highly metastatic behavior, like their human counterparts. Finally, we provide evidence that patient-derived pleomorphic sarcoma cells are inclined to spontaneous cell fusion. The resulting hybrids also gain in aggressiveness, exhibiting superior growth capacity in mouse models. Altogether, these results indicate that cell fusion has the potential to promote cancer progression by increasing growth and/or metastatic capacities, regardless of the nature of the companion cell. Moreover, such events likely occur upon sarcoma development, paving the way for better understanding of the biology, and aggressiveness of these tumors.
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Transição Epitelial-Mesenquimal , Genoma Humano , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Sarcoma/metabolismo , Animais , Fusão Celular , Linhagem Celular Tumoral , Humanos , Células-Tronco Mesenquimais/patologia , Camundongos , Metástase Neoplásica , Células-Tronco Neoplásicas/patologia , Sarcoma/patologiaRESUMO
Soft-tissue sarcomas (STS) are rare tumors whose oncogenesis remains unknown and for which no common therapeutic target has yet been identified. Analysis of 318 STS by CGH array evidenced a frequent deletion affecting the DMD gene (encoding dystrophin isoforms) in 16.5% of STS, including sarcomas with complex genomics, gastrointestinal tumors (GIST), and synovial sarcomas (SS). These deletions are significantly associated with metastatic progression, thus suggesting the role of DMD downregulation in the acquisition of aggressive phenotypes. We observed that targeted deletions of DMD were restricted to the 5' region of the gene, which is responsible for the transcription of Dp427. Analysis of STS tumors and cell lines by RNA sequencing revealed that only the Dp71 isoform was widely expressed. Dp427 depletion had no effect on cell growth or migration. However, Dp71 inhibition by shRNA dramatically reduced the cell proliferation and clonogenicity of three STS cell lines, likely by altering the cell cycle progression through the G2/M-phase. Our work demonstrates that DMD deletions are not restricted to myogenic tumors and could be used as a biomarker for metastatic evolution in STS. Dp71 seems to play an essential role in tumor growth, thus providing a potential target for future STS treatments.
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Half of soft-tissue sarcomas are tumors with complex genomics, which display no specific genetic alterations and respond poorly to treatment. It is therefore necessary to find new therapeutic targets for these sarcomas. Despite genetic heterogeneity across samples, oncogenesis may be driven by common pathway alterations. Therefore, genomic and transcriptomic profiles of 106 sarcomas with complex genomics were analyzed to identify common pathways with altered genes. This brought out a gene belonging to the "cell cycle" biological pathway, RCBTB1 (RCC1 And BTB Domain Containing Protein 1), which is lost and downregulated in 62.5% of metastatic tumors against 34% of non-metastatic tumors. A retrospective study of three sarcoma cohorts revealed that low RCBTB1 expression is prognostic for metastatic progression, specifically in patients that received chemotherapy. In vitro and in vivo, RCBTB1 overexpression in leiomyosarcoma cells specifically sensitized to docetaxel-induced apoptosis. This was associated with increased mitotic rate in vitro and higher growth rate of xenografts. By contrast, RCBTB1 inhibition decreased cell proliferation and protected sarcoma cells from apoptosis induced by docetaxel. Collectively, these data evidenced that RCBTB1 is frequently deleted in sarcomas with complex genomics and that its downregulation is associated with a higher risk of developing metastasis for patients receiving chemotherapy, likely due to their higher resistance to docetaxel.
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Oncogenesis is considered to result from chromosomal instability, in addition to oncogene and tumor-suppressor alterations. Intermediate to aneuploidy and chromosomal instability, genome doubling is a frequent event in tumor development but the mechanisms driving tetraploidization and its impact remain unexplored. Cell fusion, one of the pathways to tetraploidy, is a physiological process involved in mesenchymal cell differentiation. Besides simple genome doubling, cell fusion results in the merging of two different genomes that can be destabilized upon proliferation. By testing whether cell fusion is involved in mesenchymal oncogenesis, we provide evidence that it induces genomic instability and mediates tumor initiation. After a latency period, the tumor emerges with the cells most suited for its development. Furthermore, hybrid tumor genomes were stabilized after this selection process and were very close to those of human pleomorphic mesenchymal tumors. Thus genome restructuring triggered by cell fusion may account for the chromosomal instability involved in oncogenesis.
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Aneuploidia , Transformação Celular Neoplásica/genética , Instabilidade Cromossômica/fisiologia , Células Híbridas/citologia , Células Híbridas/metabolismo , Neoplasias/genética , Animais , Fusão Celular , Células Cultivadas , Instabilidade Genômica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Neoplasias/patologia , TetraploidiaRESUMO
RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.
Assuntos
RNA Polimerase III/metabolismo , Transcrição Gênica , Animais , Transformação Celular Neoplásica , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Modelos Biológicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase III/genética , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Telomerase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
PURPOSE: Despite various differences, nontranslocation-related sarcomas (e.g., comprising undifferentiated pleomorphic sarcoma, leiomyosarcoma, myxofibrosarcoma) are unified by their complex genetics. Extensive analysis of the tumor genome using molecular cytogenetic approaches showed many chromosomal gains, losses, and translocations per cell. Genomic quantitative alterations and expression variations have been extensively studied by adapted high-throughput approaches, yet translocations still remained unscreened. We therefore analyzed 117 nontranslocation-related sarcomas by RNA sequencing to identify fusion genes. EXPERIMENTAL DESIGN: We performed RNA sequencing and applied a bioinformatics pipeline dedicated to the detection of fusion transcripts. RT-PCR and Sanger sequencing were then applied to validate predictions and to search for recurrence and specificity. RESULTS: Among the 6,772 predicted fusion genes, 420 were in-frame. One recurrent rearrangement, consistently involving TRIO with various partners, was identified in 5.1% of cases. TRIO translocations are either intrachromosomal with TERT or interchromosomal with LINC01504 or ZNF558 Our results suggest that all translocations led to a truncated TRIO protein either directly or indirectly by alternative splicing. TRIO rearrangement is associated with a modified transcriptomic program to immunity/inflammation, proliferation and migration, and an increase in proliferation. CONCLUSIONS: TRIO fusions have been identified in four different sarcoma histotypes, likely meaning that they are not related to a primary oncogenic event but rather to a secondary one implicated in tumor progression. Moreover, they appear to be specific to nontranslocation-related sarcomas, as no such rearrangement was identified in sarcomas with simple genetics. More cases could lead to a significant association of these fusions to a specific clinical behavior. Clin Cancer Res; 23(3); 857-67. ©2016 AACR.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Serina-Treonina Quinases/genética , Sarcoma/genética , Idoso , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Feminino , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/fisiologia , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/fisiologia , Interferência de RNA , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Sarcoma/classificação , Sarcoma/metabolismo , Sarcoma/patologia , Análise de Sequência de RNA , Telomerase/genética , Telomerase/metabolismo , Translocação GenéticaRESUMO
BACKGROUND: Prognosis of metastatic outcome in soft tissue sarcomas is an important clinical challenge since these tumours can be very aggressive (up to 50% of recurring events). A gene expression signature, Complexity INdex in SARComas (CINSARC), has been identified as a better prognostic factor compared to the current international grading system defined by the Fédération Nationale des Centres de Lutte Contre le Cancer. Since CINSARC has been established on frozen tumours analysed by microarrays, we were interested in evaluating its prognostic capacity using next generation sequencing (NGS) on formalin-fixed, paraffin-embedded (FFPE) blocks to better fit laboratory practices. METHODS: Metastatic-free survivals (training/validation approach with independent datasets) and agreement values in classification groups were evaluated. Also, RNA degradation threshold has been established for FFPE blocks and differences in gene expression due to RNA degradation were measured. RESULTS: CINSARC remains a strong prognostic factor for metastatic outcome in both microarray and RNA-seq technologies (P < 0.05), with similar risk-group classifications (77%). We defined quality threshold to process degraded RNA extracted from FFPE blocks and measured similar classifications with frozen tumours (88%). CONCLUSION: These results demonstrate that CINSARC is a platform and material independent prognostic signature for metastatic outcome in various sarcomas. This result opens access to metastatic prognostication in sarcomas through NGS analysis on both frozen and FFPE tumours via the CINSARC signature.
Assuntos
RNA Neoplásico/genética , Sarcoma/genética , Análise de Sequência de RNA/métodos , Neoplasias de Tecidos Moles/genética , Idoso , Intervalo Livre de Doença , Perfilação da Expressão Gênica/normas , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estimativa de Kaplan-Meier , Análise em Microsséries/métodos , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Sarcoma/mortalidade , Neoplasias de Tecidos Moles/mortalidadeRESUMO
Nutlin inhibits TP53-MDM2 interaction and is under investigation in soft-tissue sarcomas (STS) and other malignancies. Molecular mechanisms of secondary resistance to nutlin in STS are unknown. We performed whole-transcriptome sequencing (RNA-seq) on three pretreatment and secondary resistant STS cell lines selected based on their high primary sensitivity to nutlin. Our data identified a subset of cancer gene mutations and ploidy variations that were positively selected following treatment, including TP53 mutations in 2 out of 3 resistant cell lines. Further, secondary resistance to nutlin was associated with deregulation of apoptosis-related genes and marked productive autophagy, the inhibition of which resulted in significant restoration of nutlin-induced cell death. Collectively, our findings argue that secondary resistance to nutlin in STS involved heterogeneous mechanisms resulting from clonal evolution and several biological pathways. Alternative dosing regimens and combination with other targeted agents are needed to achieve successful development of nutlin in the clinical setting.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Imidazóis/farmacologia , Sarcoma/tratamento farmacológico , Sarcoma/patologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Células Clonais/patologia , Perfilação da Expressão Gênica , Humanos , Mutação , Ploidias , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirrolidinas/farmacologia , Sarcoma/genética , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , para-Aminobenzoatos/farmacologiaRESUMO
PURPOSE: Imatinib mesylate is the front-line targeted therapy for gastrointestinal stromal tumours (GISTs). Patient's eligibility to adjuvant imatinib after primary tumour resection is currently based on histological and clinical risk assessment. While therapeutic options are clear for the very-low, low and high-risk subpopulations, no standard is actually available for the tumours classified as intermediate. Since we recently validated genomic index (GI), a measure of the level of genomic alterations, as a strong predictor of clinical outcome in GIST, we asked whether it could also represent a novel prognostic factor for the intermediate subgroup. EXPERIMENTAL DESIGN: 82 intermediate risk patients were selected based on the Armed Forces Institute of Pathology (AFIP) classification for genomic profiling. RESULTS: Data revealed that even if studied samples generally harboured a combination of the typical genetic aberrations found in GIST, i.e. 1p, 14q 22q deletions and frequently lost CDKN2A locus on chromosome 9, they profoundly differed from each other on the total number of genomic changes and GI value. Kaplan-Meier analyses of metastatic-free survival unveiled that stratification of the tumours by the GI value at a cutoff of 10 separated the good from the poor prognosis patients, proven that metastatic-risk in GIST intermediate patients is strongly associated with high GI values and genome complexity. CONCLUSION: GI is validated here as a robust marker to predict intermediate-GIST clinical outcome. Applicable in numerous Pathology Laboratories already using array comparative genomic hybridisation (CGH) with formalin-fixed paraffin-embedded (FFPE) samples, this assay presently stands as an efficient tool for the clinical management of intermediate GIST-patients.