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IL-1 family members have multiple pleiotropic functions affecting various tissues and cells, including the regulation of the immune response, hematopoietic homeostasis, bone remodeling, neuronal physiology, and synaptic plasticity. Many of these activities are involved in various pathological processes and immunological disorders, including tumor initiation and progression. Indeed, IL-1 family members have been described to contribute to shaping the tumor microenvironment (TME), determining immune evasion and drug resistance, and to sustain tumor aggressiveness and metastasis. This review addresses the role of IL-1 family members in bone sarcomas, particularly the highly metastatic osteosarcoma (OS) and Ewing sarcoma (EWS), and discusses the IL-1-family-related mechanisms that play a role in bone metastasis development. We also consider the therapeutic implications of targeting IL-1 family members, which have been proposed as (i) relevant targets for anti-tumor and anti-metastatic drugs; (ii) immune checkpoints for immune suppression; and (iii) potential antigens for immunotherapy.
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Neoplasias Óseas , Osteosarcoma , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/patología , Neoplasias Óseas/patología , Familia , Interleucina-1 , Microambiente TumoralRESUMEN
Ewing sarcoma (EWS) is the second most common pediatric bone tumor. The EWS tumor microenvironment is largely recognized as immune-cold, with macrophages being the most abundant immune cells and their presence associated with worse patient prognosis. Expression of CD99 is a hallmark of EWS cells, and its targeting induces inhibition of EWS tumor growth through a poorly understood mechanism. In this study, we analyzed CD99 expression and functions on macrophages and investigated whether the concomitant targeting of CD99 on both tumor and macrophages could explain the inhibitory effect of this approach against EWS. Targeting CD99 on EWS cells downregulated expression of the "don't eat-me" CD47 molecule but increased levels of the "eat-me" phosphatidyl serine and calreticulin molecules on the outer leaflet of the tumor cell membrane, triggering phagocytosis and digestion of EWS cells by macrophages. In addition, CD99 ligation induced reprogramming of undifferentiated M0 macrophages and M2-like macrophages toward the inflammatory M1-like phenotype. These events resulted in the inhibition of EWS tumor growth. Thus, this study reveals what we believe to be a previously unrecognized function of CD99, which engenders a virtuous circle that delivers intrinsic cell death signals to EWS cells, favors tumor cell phagocytosis by macrophages, and promotes the expression of various molecules and cytokines, which are pro-inflammatory and usually associated with tumor regression. This raises the possibility that CD99 may be involved in boosting the antitumor activity of macrophages.
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Neoplasias Óseas , Sarcoma de Ewing , Humanos , Niño , Sarcoma de Ewing/genética , Muerte Celular , Línea Celular Tumoral , Macrófagos/metabolismo , Microambiente Tumoral , Antígeno 12E7RESUMEN
Synovial sarcoma (SyS) is a rare aggressive soft tissue sarcoma carrying the chromosomal translocation t(X;18), encoding the fusion transcript SS18::SSX. The fusion oncoprotein interacts with both BAF enhancer complexes and polycomb repressor complexes, resulting in genome-wide epigenetic perturbations and a unique altered genetic signature. Over 80% of the patients are initially diagnosed with localized disease and have a 5-year survival rate of 70-80%, but metastatic relapse occurs in 50% of the cases. Advanced, unresectable, or metastatic disease has a 5-year survival rate below 10%, representing a critical issue. This review summarizes the molecular mechanisms behind SyS and illustrates current treatments in front line, second line, and beyond settings. We analyze the use of immune check point inhibitors (ICI) in SyS that do not behave as an ICI-sensitive tumor, claiming the need for predictive genetic signatures and tumor immune microenvironment biomarkers. We highlight the clinical translation of innovative technologies, such as proteolysis targeting chimera (PROTAC) protein degraders or adoptive transfer of engineered immune cells. Adoptive cell transfer of engineered T-cell receptor cells targeting selected cancer/testis antigens has shown promising results against metastatic SyS in early clinical trials and further improvements are awaited from refinements involving immune cell engineering and tumor immune microenvironment enhancement.
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The biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 µg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research.
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PURPOSE: The relevance of the subfamily A members of ATP-binding cassette (ABCA) transporters as biomarkers of risk and response is emerging in different tumors, but their mechanisms of action have only been partially defined. In this work, we investigated their role in Ewing sarcoma (EWS), a pediatric cancer with unmet clinical issues. METHODS: The expression of ABC members was evaluated by RT-qPCR in patients with localized EWS. The correlation with clinical outcome was established in different datasets using univariate and multivariate statistical methods. Functional studies were conducted in cell lines from patient-derived xenografts (PDXs) using gain- or loss-of-function approaches. The impact of intracellular cholesterol levels and cholesterol lowering drugs on malignant parameters was considered. RESULTS: We found that ABCA6, which is usually poorly expressed in EWS, when upregulated became a prognostic factor of a favorable outcome in patients. Mechanistically, high expression of ABCA6 impaired cell migration and increased cell chemosensitivity by diminishing the intracellular levels of cholesterol and by constitutive IGF1R/AKT/mTOR expression/activation. Accordingly, while exposure of cells to exogenous cholesterol increased AKT/mTOR activation, the cholesterol lowering drug simvastatin inhibited IGF1R/AKT/mTOR signaling and prevented Ser166 phosphorylation of MDM2. This, in turn, favored p53 activation and enhanced pro-apoptotic effects of doxorubicin. CONCLUSIONS: Our study reveals that ABCA6 acts as tumor suppressor in EWS cells via cholesterol-mediated inhibition of IGF1R/AKT/MDM2 signaling, which promotes the pro-apoptotic effects of doxorubicin and reduces cell migration. Our findings also support a role of ABCA6 as biomarker of EWS progression and sustains its assessment for a more rational use of statins as adjuvant drugs.
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Transportadoras de Casetes de Unión a ATP , Sarcoma de Ewing , Niño , Humanos , Transportadoras de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Colesterol , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Receptor IGF Tipo 1 , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Serina-Treonina Quinasas TOR/metabolismo , AnimalesRESUMEN
Background: Giant cell tumors of bone (GCTB) are osteolytic tumors. Denosumab, a RANK-L inhibitor, is approved for GCTB. Data on serum bone turnover marker (sBTM) changes are lacking. We present a phase II correlative study on sBTMs in GCTB patients treated with denosumab. Methods: All GCTB patients receiving denosumab within a multicentre, open-label, phase 2 study were enrolled. Serum levels of carboxyterminal-crosslinked-telopeptide of type I collagen (s-CTX), alkaline phosphatase (ALP), bone-alkaline phosphatase (bALP), parathyroid hormone (sPTH), and osteocalcin (OCN) were prospectively assessed (baseline, T0, 3 months, T1, 6 months, T2). The primary endpoint was assessment of sBTM changes after denosumab; the secondary endpoints were disease-free survival (DFS) and sBTM correlation. Results: In 54 cases, sBTMs decreased during denosumab treatment except for sPTH. With a median follow-up of 59 months, 3-year DFS was 65% (%CI 52−79), with a significantly worse outcome for patients with high (≥500 UI/mL) s-CTX at baseline, as compared to low s-CTX (<500 UI/mL) (3-year DFS for high CTX 45% (95%CI 23−67) vs. 75% (95%CI 59−91) for low s-CTX. Higher median ALP and s-CTX were found for patients with tumor size ≥ 5 cm (p = 0.0512; p = 0.0589). Conclusion: Denosumab induces ALP/OCN and s-CTX reduction. High baseline s-CTX identifies a group of patients at higher risk of progression of the disease.
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DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor in children and adolescents. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming and the DNMT1 enzyme has been demonstrated to have an important role in both maintaining the epigenome and controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor proliferation through a mechanism that is independent on DNA methylation. Depletion of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 phase, and DNA damage, as revealed by the induction of γH2AX foci. These effects elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 mutated cells, persistent micronuclei and increased DNA instability was observed. Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 as an adjuvant agent in treating EWS.
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Sarcoma de Ewing , Adolescente , Benzamidas , Línea Celular Tumoral , Proliferación Celular , Niño , ADN/metabolismo , Daño del ADN , Metilación de ADN , Inhibidores Enzimáticos/farmacología , Humanos , Pirimidinas , Quinolinas , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/farmacologíaRESUMEN
Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness. Accordingly, in experimental in vitro models, low lamin A expression correlated with enhanced cell migration and invasiveness and, in vivo, with an increased metastatic load. At the molecular level, this condition was linked to altered expression and anchorage of nuclear envelope proteins and increased nuclear retention of YAP/TAZ, a mechanosignaling effector. Conversely, overexpression of lamin A rescued LINC complex organization, thus reducing YAP/TAZ nuclear recruitment and preventing cell invasiveness. These effects were also obtained through modulation of lamin A maturation by a statin-based pharmacological treatment that further elicited a more differentiated phenotype in EWS cells. These results demonstrate that drugs inducing nuclear envelope remodeling could be exploited to improve therapeutic strategies for EWS.
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Membrana Nuclear , Sarcoma de Ewing , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismoRESUMEN
Capicua-double homeobox 4 (CIC-DUX4)-rearranged sarcomas (CDS) are extremely rare, highly aggressive primary sarcomas that represent a major therapeutic challenge. Patients are treated according to Ewing sarcoma protocols, but CDS-specific therapies are strongly needed. In this study, RNA sequencing was performed on patient samples to identify a selective signature that differentiates CDS from Ewing sarcoma and other fusion-driven sarcomas. This signature was used to validate the representativeness of newly generated CDS experimental models-patient-derived xenografts (PDX) and PDX-derived cell lines-and to identify specific therapeutic vulnerabilities. Annotation analysis of differentially expressed genes and molecular gene validation highlighted an HMGA2/IGF2BP/IGF2/IGF1R/AKT/mTOR axis that characterizes CDS and renders the tumors particularly sensitive to combined treatments with trabectedin and PI3K/mTOR inhibitors. Trabectedin inhibited IGF2BP/IGF2/IGF1R activity, but dual inhibition of the PI3K and mTOR pathways was required to completely dampen downstream signaling mediators. Proof-of-principle efficacy for the combination of the dual AKT/mTOR inhibitor NVP-BEZ235 (dactolisib) with trabectedin was obtained in vitro and in vivo using CDS PDX-derived cell lines, demonstrating a strong inhibition of local tumor growth and multiorgan metastasis. Overall, the development of representative experimental models (PDXs and PDX-derived cell lines) has helped to identify the unique sensitivity of the CDS to AKT/mTOR inhibitors and trabectedin, revealing a mechanism-based therapeutic strategy to fight this lethal cancer. SIGNIFICANCE: This study identifies altered HMGA2/IGF2BP/IGF2 signaling in CIC-DUX4 sarcomas and provides proof of principle for combination therapy with trabectedin and AKT/mTOR dual inhibitors to specifically combat the disease.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/genética , Sarcoma/tratamiento farmacológico , Neoplasias de los Tejidos Blandos/tratamiento farmacológico , Animales , Línea Celular Tumoral , Redes Reguladoras de Genes , Humanos , Estimación de Kaplan-Meier , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteínas de Fusión Oncogénica/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Sarcoma/genética , Sarcoma/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/metabolismo , Trabectedina/administración & dosificación , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Ewing sarcoma, a highly aggressive pediatric tumor, is driven by EWS-FLI1, an oncogenic transcription factor that remodels the tumor genetic landscape. Epigenetic mechanisms play a pivotal role in Ewing sarcoma pathogenesis, and the therapeutic value of compounds targeting epigenetic pathways is being identified in preclinical models. Here, we showed that modulation of CD99, a cell surface molecule highly expressed in Ewing sarcoma cells, may alter transcriptional dysregulation in Ewing sarcoma through control of the zyxin-GLI1 axis. Zyxin is transcriptionally repressed, but GLI1 expression is maintained by EWS-FLI1. We demonstrated that targeting CD99 with antibodies, including the human diabody C7, or genetically inhibiting CD99 is sufficient to increase zyxin expression and induce its dynamic nuclear accumulation. Nuclear zyxin functionally affects GLI1, inhibiting targets such as NKX2-2, cyclin D1, and PTCH1 and upregulating GAS1, a tumor suppressor protein negatively regulated by SHH/GLI1 signaling. We used a battery of functional assays to demonstrate (i) the relationship between CD99/zyxin and tumor cell growth/migration and (ii) how CD99 deprivation from the Ewing sarcoma cell surface is sufficient to specifically affect the expression of some crucial EWS-FLI1 targets, both in vitro and in vivo, even in the presence of EWS-FLI1. This article reveals that the CD99/zyxin/GLI1 axis is promising therapeutic target for reducing Ewing sarcoma malignancy.
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Antígeno 12E7 , Proteínas de Fusión Oncogénica , Oncogenes , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Sarcoma de Ewing , Proteína con Dedos de Zinc GLI1 , Zixina , Animales , Humanos , Ratones , Antígeno 12E7/metabolismo , Ratones Desnudos , Proteínas de Fusión Oncogénica/metabolismo , Oncogenes/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Transfección , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Zixina/genéticaRESUMEN
The ATP-binding cassette (ABC) transporter superfamily consists of several proteins with a wide repertoire of functions. Under physiological conditions, ABC transporters are involved in cellular trafficking of hormones, lipids, ions, xenobiotics, and several other molecules, including a broad spectrum of chemical substrates and chemotherapeutic drugs. In cancers, ABC transporters have been intensely studied over the past decades, mostly for their involvement in the multidrug resistance (MDR) phenotype. This review provides an overview of ABC transporters, both related and unrelated to MDR, which have been studied in osteosarcoma and Ewing's sarcoma. Since different backbone drugs used in first-line or rescue chemotherapy for these two rare bone sarcomas are substrates of ABC transporters, this review particularly focused on studies that have provided findings that have been either translated to clinical practice or have indicated new candidate therapeutic targets; however, findings obtained from ABC transporters that were not directly involved in drug resistance were also discussed, in order to provide a more complete overview of the biological impacts of these molecules in osteosarcoma and Ewing's sarcoma. Finally, therapeutic strategies and agents aimed to circumvent ABC-mediated chemoresistance were discussed to provide future perspectives about possible treatment improvements of these neoplasms.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Osteosarcoma/tratamiento farmacológico , Sarcoma de Ewing/tratamiento farmacológico , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Humanos , Osteosarcoma/metabolismo , Osteosarcoma/patología , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologíaRESUMEN
Osteosarcoma (OS) is a rare malignant primary tumor of mesenchymal origin affecting bone. It is characterized by a complex genotype, mainly due to the high frequency of chromothripsis, which leads to multiple somatic copy number alterations and structural rearrangements. Any effort to design genome-driven therapies must therefore consider such high inter- and intra-tumor heterogeneity. Therefore, many laboratories and international networks are developing and sharing OS patient-derived xenografts (OS PDX) to broaden the availability of models that reproduce OS complex clinical heterogeneity. OS PDXs, and new cell lines derived from PDXs, faithfully preserve tumor heterogeneity, genetic, and epigenetic features and are thus valuable tools for predicting drug responses. Here, we review recent achievements concerning OS PDXs, summarizing the methods used to obtain ectopic and orthotopic xenografts and to fully characterize these models. The availability of OS PDXs across the many international PDX platforms and their possible use in PDX clinical trials are also described. We recommend the coupling of next-generation sequencing (NGS) data analysis with functional studies in OS PDXs, as well as the setup of OS PDX clinical trials and co-clinical trials, to enhance the predictive power of experimental evidence and to accelerate the clinical translation of effective genome-guided therapies for this aggressive disease.
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Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Xenoinjertos/inmunología , Osteosarcoma/inmunología , Animales , Humanos , Osteosarcoma/genética , Trasplante Heterólogo/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Ewing sarcoma (EWS) is a rare malignant pediatric tumor and patient derived xenografts (PDXs) could represent a possibility to increase the number of available models to study this disease. Compared to cell derived xenografts (CDX), PDXs are reported to better recapitulate tumor microenvironment, heterogeneity, genetic and epigenetic features and are considered reliable models for their better predictive value when comparing preclinical efficacy and treatment response in patients. In this chapter, we extensively describe a method for generating Ewing sarcoma PDX models, for their validation and molecular characterization.
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Neoplasias Óseas/patología , Modelos Animales de Enfermedad , Xenoinjertos , Sarcoma de Ewing/patología , Animales , Biopsia , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microambiente TumoralRESUMEN
Ewing sarcoma (EWS) is the second most common bone and soft tissue-associated malignancy in children and young adults. It is driven by the fusion oncogene EWS/FLI1 and characterized by rapid growth and early metastasis. We have previously discovered that the mRNA binding protein IGF2BP3 constitutes an important biomarker for EWS as high expression of IGF2BP3 in primary tumors predicts poor prognosis of EWS patients. We additionally demonstrated that IGF2BP3 enhances anchorage-independent growth and migration of EWS cells suggesting that IGF2BP3 might work as molecular driver and predictor of EWS progression. The aim of this study was to further define the role of IGF2BP3 in EWS progression. We demonstrated that high IGF2BP3 mRNA expression levels correlated with EWS metastasis and disease progression in well-characterized EWS tumor specimens. EWS tumors with high IGF2BP3 levels were characterized by a specific gene signature enriched in chemokine-mediated signaling pathways. We also discovered that IGF2BP3 regulated the expression of CXCR4 through CD164. Significantly, CD164 and CXCR4 colocalized at the plasma membrane of EWS cells upon CXCL12 stimulation. We further demonstrated that IGF2BP3, CD164, and CXCR4 expression levels correlated in clinical samples and the IGF2BP3/CD164/CXCR4 signaling pathway promoted motility of EWS cells in response to CXCL12 and under hypoxia conditions. The data presented identified CD164 and CXCR4 as novel IGF2BP3 downstream functional effectors indicating that the IGF2BP3/CD164/CXCR4 oncogenic axis may work as critical modulator of EWS aggressiveness. In addition, IGF2BP3, CD164, and CXCR4 expression levels may constitute a novel biomarker panel predictive of EWS progression.
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Appropriate tools for monitoring sarcoma progression are still limited. The aim of the present study was to investigate the value of miR-34a-5p (miR34a) as a circulating biomarker to follow disease progression and measure the therapeutic response. Stable forced re-expression of miR34a in Ewing sarcoma (EWS) cells significantly limited tumor growth in mice. Absolute quantification of miR34a in the plasma of mice and 31 patients showed that high levels of this miRNA inversely correlated with tumor volume. In addition, miR34a expression was higher in the blood of localized EWS patients than in the blood of metastatic EWS patients. In 12 patients, we followed miR34a expression during preoperative chemotherapy. While there was no variation in the blood miR34a levels in metastatic patients at the time of diagnosis or after the last cycle of preoperative chemotherapy, there was an increase in the circulating miR34a levels in patients with localized tumors. The three patients with the highest fold-increase in the miR levels did not show evidence of metastasis. Although this analysis should be extended to a larger cohort of patients, these findings imply that detection of the miR34a levels in the blood of EWS patients may assist with the clinical management of EWS.
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BACKGROUND: The treatment of metastatic osteosarcoma (OS) remains a challenge for oncologists, and novel therapeutic strategies are urgently needed. An understanding of the pathways that regulate OS dissemination is required for the design of novel treatment approaches. We recently identified Rho-associated coiled-coil containing protein kinase 2 (ROCK2) as a crucial driver of OS cell migration. In this study, we explored the impact of ROCK2 disruption on the metastatic capabilities of OS cells and analyzed its functional relationship with Yes-associated protein-1 (YAP), the main transcriptional mediator of mechanotransduction signaling. METHODS: The effects of ROCK2 depletion on metastasis were studied in NOD Scid gamma (NSG) mice injected with U-2OS cells in which ROCK2 expression had been stably silenced. Functional studies were performed in vitro in human U-2OS cells and in three novel cell lines derived from patient-derived xenografts (PDXs) by using standard methods to evaluate malignancy parameters and signaling transduction. The nuclear immunostaining of YAP and the evaluation of its downstream targets Cysteine Rich Angiogenic Inducer 6, Connective Tissue Growth Factor and Cyclin D1 by quantitative PCR were performed to analyze YAP activity. The effect of the expression and activity of ROCK2 and YAP on tumor progression was analyzed in 175 OS primary tumors. RESULTS: The silencing of ROCK2 markedly reduced tumor growth and completely abolished the metastatic ability of U-2OS cells. The depletion of ROCK2, either by pharmacological inhibition or silencing, induced a dose- and time-dependent reduction in the nuclear expression and transcriptional activity of YAP. The nuclear expression of YAP was observed in 80/175 (46%) tumor samples and was significantly correlated with worse patient prognosis and a higher likelihood of metastasis and death. The use of verteporfin, a molecule that specifically inhibits the TEAD-YAP association, remarkably impaired the growth and migration of OS cells in vitro. Moreover to inhibiting YAP activity, our findings indicate that verteporfin also affects the ROCK2 protein and its functions. CONCLUSIONS: We describe the functional connection between ROCK2 and YAP in the regulation of OS cell migration and metastasis formation. These data provide support for the use of verteporfin as a possible therapeutic option to prevent OS cell dissemination.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias Óseas/metabolismo , Osteosarcoma/metabolismo , Factores de Transcripción/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias Óseas/terapia , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Niño , Modelos Animales de Enfermedad , Femenino , Xenoinjertos , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Ratones , Osteosarcoma/genética , Osteosarcoma/patología , Osteosarcoma/terapia , Pronóstico , Unión Proteica , Interferencia de ARN , Factores de Transcripción/genética , Verteporfina/farmacología , Proteínas Señalizadoras YAP , Quinasas Asociadas a rho/genéticaRESUMEN
Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches.
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Antineoplásicos/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Osteosarcoma/tratamiento farmacológico , Sarcoma de Ewing/tratamiento farmacológico , Antígeno 12E7/inmunología , Animales , Anticuerpos/uso terapéutico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Irinotecán/uso terapéutico , Ratones , Ratones Endogámicos NOD , Ratones SCID , Osteosarcoma/metabolismo , Osteosarcoma/patología , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: DNA methyltransferases (DNMTs) are epigenetic enzymes involved in embryonic development, cell differentiation, epithelial to mesenchymal transition, and control of gene expression, whose overexpression or enhanced catalytic activity has been widely reported in cancer initiation and progression. To date, two DNMT inhibitors (DNMTi), 5-azacytidine (5-AZA) and 5-aza-2'-deoxycytidine (DAC), are approved for the treatment of myelodysplastic syndromes and acute myeloid leukemia. Nevertheless, they are chemically instable and quite toxic for healthy cells; thus, the discovery of novel DNMTi is urgent. RESULTS: Here, we report the identification of a new quinoline-based molecule, MC3353, as a non-nucleoside inhibitor and downregulator of DNMT. This compound was able, in promoter demethylating assays, to induce enhanced green fluorescence protein (EGFP) gene expression in HCT116 cells and transcription in a cytomegalovirus (CMV) promoter-driven luciferase reporter system in KG-1 cells. Moreover, MC3353 displayed a strong antiproliferative activity when tested on HCT116 colon cancer cells after 48 h of treatment at 0.5 µM. At higher doses, this compound provided a cytotoxic effect in double DNMT knockout HCT116 cells. MC3353 was also screened on a different panel of cancer cells (KG-1 and U-937 acute myeloid leukemia, RAJI Burkitt's lymphoma, PC-3 prostate cancer, and MDA-MB-231 breast cancer), where it arrested cell proliferation and reduced viability after 48 h of treatment with IC50 values ranging from 0.3 to 0.9 µM. Compared to healthy cell models, MC3353 induced apoptosis (e.g., U-937 and KG-1 cells) or necrosis (e.g., RAJI cells) at lower concentrations. Importantly, together with the main DNMT3A enzyme inhibition, MC3353 was also able to downregulate the DNMT3A protein level in selected HCT116 and PC-3 cell lines. Additionally, this compound provided impairment of the epithelial-to-mesenchymal transition (EMT) by inducing E-cadherin while reducing matrix metalloproteinase (MMP2) mRNA and protein levels in PC-3 and HCT116 cells. Last, tested on a panel of primary osteosarcoma cell lines, MC3353 markedly inhibited cell growth with low single-digit micromolar IC50 ranging from 1.1 to 2.4 µM. Interestingly, in Saos-2 osteosarcoma cells, MC3353 induced both expression of genes and mineralized the matrix as evidence of osteosarcoma to osteoblast differentiation. CONCLUSIONS: The present work describes MC3353 as a novel DNMTi displaying a stronger in cell demethylating ability than both 5-AZA and DAC, providing re-activation of the silenced ubiquitin C-terminal hydrolase L1 (UCHL1) gene. MC3353 displayed dose- and time-dependent antiproliferative activity in several cancer cell types, inducing cell death and affecting EMT through E-cadherin and MMP2 modulation. In addition, this compound proved efficacy even in primary osteosarcoma cell models, through the modulation of genes involved in osteoblast differentiation.