RESUMO
Bone sarcomas are malignant tumors of mesenchymal origin. Complete surgical resection is the cornerstone of multidisciplinary treatment. However, advanced, unresectable forms remain incurable. A crucial step towards addressing this challenge involves comprehending the molecular mechanisms underpinning tumor progression and metastasis, laying the groundwork for innovative precision medicine-based interventions. We previously showed that tyrosine kinase receptor Ephrin Type-A Receptor 2 (EphA2) is overexpressed in bone sarcomas. EphA2 is a key oncofetal protein implicated in metastasis, self-renewal, and chemoresistance. Molecular, genetic, biochemical, and pharmacological approaches have been developed to target EphA2 and its signaling pathway aiming to interfere with its tumor-promoting effects or as a carrier for drug delivery. This review synthesizes the main functions of EphA2 and their relevance in bone sarcomas, providing strategies devised to leverage this receptor for diagnostic and therapeutic purposes, with a focus on its applicability in the three most common bone sarcoma histotypes: osteosarcoma, chondrosarcoma, and Ewing sarcoma.
Assuntos
Neoplasias Ósseas , Receptor EphA2 , Transdução de Sinais , Humanos , Receptor EphA2/metabolismo , Receptor EphA2/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Animais , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Osteossarcoma/genética , Terapia de Alvo Molecular , Sarcoma/metabolismo , Sarcoma/genética , Sarcoma/patologiaRESUMO
BACKGROUND: Osteosarcoma (OS) survival rates and outcome have not improved in 50 years since the advent of modern chemotherapeutics. Thus, there is a critical need for an improved understanding of the tumor microenvironment to identify better therapies. Extracellular matrix (ECM) deposition and hypoxia are known to abrogate the efficacy of various chemical and cell-based therapeutics. Here, we aim to mechanistically investigate the combinatorial effects of hypoxia and matrix deposition with the use of OS spheroids. METHODS: We use two murine OS cell lines with differential metastatic potential to form spheroids. We form spheroids of two sizes, use ascorbate-2-phosphate supplementation to enhance ECM deposition, and study cell response under standard (21% O2) and physiologic (5% O2) oxygen tensions. Finally, we examine chemotherapeutic responses to doxorubicin treatment. RESULTS: ECM production and oxygen tension are key determinants of spheroid size through cell organization based on nutrient and oxygen distribution. Interestingly, highly metastatic OS is more susceptible to chemotherapeutics compared to less metastatic OS when matrix production increases. Together, these data suggest that dynamic interactions between ECM production and oxygen diffusion may result in distinct chemotherapeutic responses despite inherent tumor aggressiveness. CONCLUSION: This work establishes OS spheroids as a valuable tool for early OS tumor formation investigation and holds potential for novel therapeutic target and prognostic indicator discovery.
Assuntos
Matriz Extracelular , Osteossarcoma , Oxigênio , Esferoides Celulares , Microambiente Tumoral , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Esferoides Celulares/efeitos dos fármacos , Matriz Extracelular/metabolismo , Animais , Camundongos , Oxigênio/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Despite advances in treatment, the prognosis of patients with osteosarcoma remains unsatisfactory, and searching for potential targets is imperative. Here, we identify N4-acetylcytidine (ac4C) acetyltransferase 10 (NAT10) as a candidate therapeutic target in osteosarcoma through functional screening. NAT10 overexpression is correlated with a poor prognosis, and NAT10 knockout inhibits osteosarcoma progression. Mechanistically, NAT10 enhances mRNA stability of activating transcription factor 4 (ATF4) through ac4C modification. ATF4 induces the transcription of asparagine synthetase (ASNS), which catalyzes asparagine (Asn) biosynthesis, facilitating osteosarcoma progression. Utilizing virtual screening, we identify paliperidone and AG-401 as potential NAT10 inhibitors, and both inhibitors are found to bind to NAT10 proteins. Inhibiting NAT10 suppresses osteosarcoma progression in vivo. Combined treatment using paliperidone and AG-401 produces synergistic inhibition for osteosarcoma in patient-derived xenograft (PDX) models. Our findings demonstrate that NAT10 facilitates osteosarcoma progression through the ATF4/ASNS/Asn axis, and pharmacological inhibition of NAT10 may be a feasible therapeutic approach for osteosarcoma.
Assuntos
Fator 4 Ativador da Transcrição , Asparagina , Aspartato-Amônia Ligase , Osteossarcoma , Humanos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Osteossarcoma/genética , Fator 4 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Linhagem Celular Tumoral , Aspartato-Amônia Ligase/metabolismo , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/antagonistas & inibidores , Camundongos , Asparagina/metabolismo , Progressão da Doença , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias Ósseas/patologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Camundongos Nus , Masculino , FemininoRESUMO
BACKGROUND/AIM: Methotrexate (MTX) resistance in osteosarcoma results in a very poor patient prognosis. We previously reported that super MTX-resistant osteosarcoma (143B-MTXSR) cells, selected from parental 143B osteosarcoma (143B-P) cells by culturing them with increasing concentrations of MTX, exhibited reduced malignancy, despite the over-expression of oncogenes. The present study explored the mechanism of reduced malignancy in the super MTX-resistant osteosarcoma cells. MATERIALS AND METHODS: Previously selected 143B-MTXSR cells which are 5,500 times more MTX resistant than parental cells, were used for this study. The status of methylated histone H3K9me3 and H3K27me3 marks was examined with western immunoblotting and compared between 143B-MTXSR and parental 143B-P cells. RESULTS: Histone H3K9me3 and H3K27me3 marks were over-expressed in 143B-MTXSR compared to 143B-P (p<0.05, p<0.01, respectively). CONCLUSION: Over-expression of histone H3K9me3 and H3K27me3 marks may be related to super-MTX resistance and to the loss of malignancy of super MTX-resistant osteosarcoma cells due to the fundamental relationship of methylation and cancer.
Assuntos
Neoplasias Ósseas , Resistencia a Medicamentos Antineoplásicos , Histonas , Metotrexato , Osteossarcoma , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Humanos , Metotrexato/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Histonas/metabolismo , Histonas/genética , Linhagem Celular Tumoral , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Metilação , Antimetabólitos Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: Osteosarcoma is a prevalent and aggressive primary malignant bone tumor affecting children and adolescents. Despite advancements in sequencing technologies, there remains a lack of reliable prognostic biomarkers and effective targeted therapies for osteosarcoma. This study focuses on identifying key prognostic genes, particularly the role of GNAS, in osteosarcoma progression. METHODS: Bioinformatics analyses were performed on osteosarcoma datasets from the Gene Expression Omnibus (GEO). Differential gene expression analysis, weighted correlation network analysis (WGCNA), and survival analysis identified potential prognostic hub genes. The expression and function of these genes were validated through immunohistochemistry and animal experiments. Specifically, the role of GNAS was investigated through siRNA-mediated knockdown in osteosarcoma cell lines and nude mice models. RESULTS: Five hub genes (PROP1, GNAS, CYP4F2, LHX3, CNGB1) were identified as significantly related to osteosarcoma prognosis. Among these, GNAS was found to be highly expressed in osteosarcoma tissues compared to normal tissues. Immunohistochemical analysis confirmed the elevated expression of GNAS in osteosarcoma samples. GNAS mutation analysis revealed a low mutation rate in osteosarcoma, suggesting its oncogenic role is independent of mutational status. Animal experiments demonstrated that knocking down GNAS significantly inhibited tumor growth and induced apoptosis in osteosarcoma cells. CONCLUSION: GNAS is highly expressed in osteosarcoma and associated with poor prognosis, acting as an oncogene in osteosarcoma progression. Targeting GNAS could be a potential therapeutic strategy for osteosarcoma. Further studies on GNAS-related signaling pathways may provide deeper insights into the molecular mechanisms driving osteosarcoma malignancy.
Assuntos
Neoplasias Ósseas , Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Camundongos Nus , Osteossarcoma , Animais , Humanos , Camundongos , Apoptose , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Cromograninas/genética , Cromograninas/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Camundongos Endogâmicos BALB C , Mutação , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The relationship between metastasis-associated protein 2 (MTA2) overexpression and tumor growth and metastasis has been extensively studied in a variety of tumor cells but not in human osteosarcoma cells. This study aims to elucidate the clinical significance, underlying molecular mechanisms, and biological functions of MTA2 in human osteosarcoma in vitro and in vivo. Our results show that MTA2 was elevated in osteosarcoma cell lines and osteosarcoma tissues and was associated with tumor stage and overall survival of osteosarcoma patients. Knockdown of MTA2 inhibited osteosarcoma cell migration and invasion by reducing the expression of urokinase-type plasminogen activator (uPA). Bioinformatic analysis demonstrated that high levels of uPA in human osteosarcoma tissues correlated positively with MTA2 expression. Furthermore, treatment with recombinant human uPA (Rh-uPA) caused significant restoration of OS cell migration and invasion in MTA2 knockdown osteosarcoma cells. We found that ERK1/2 depletion increased the expression of uPA, facilitating osteosarcoma cell migration and invasion. Finally, MTA2 depletion significantly reduced tumor metastasis and the formation of lung nodules in vivo. Overall, our study suggests that MTA2 knockdown suppresses osteosarcoma cell metastasis by decreasing uPA expression via ERK signaling. This finding provides new insight into potential treatment strategies against osteosarcoma metastasis by targeting MTA2.
Assuntos
Neoplasias Ósseas , Movimento Celular , Técnicas de Silenciamento de Genes , Histona Desacetilases , Osteossarcoma , Proteínas Repressoras , Ativador de Plasminogênio Tipo Uroquinase , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Humanos , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Linhagem Celular Tumoral , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Movimento Celular/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Animais , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Masculino , Feminino , Camundongos , Regulação Neoplásica da Expressão Gênica , Invasividade Neoplásica/genética , Metástase Neoplásica , Camundongos Nus , Sistema de Sinalização das MAP Quinases/genéticaRESUMO
Osteosarcoma is the most prevalent form of primary malignant bone tumor, primarily affecting children and adolescents. The nerve growth factors (NGF) referred to as neurotrophins have been associated with cancer-induced bone pain; however, the role of NGF in osteosarcoma has yet to be elucidated. In osteosarcoma samples from the Genomic Data Commons data portal, we detected higher levels of NGF and M2 macrophage markers, but not M1 macrophage markers. In cellular experiments, NGF-stimulated osteosarcoma conditional medium was shown to facilitate macrophage polarization from the M0 to the M2 phenotype. NGF also enhanced VCAM-1-dependent monocyte adhesion within the osteosarcoma microenvironment by down-regulating miR-513c-5p levels through the FAK and c-Src cascades. In in vivo xenograft models, the overexpression of NGF was shown to enhance tumor growth, while the oral administration of the TrK inhibitor larotrectinib markedly antagonized NGF-promoted M2 macrophage expression and tumor progression. These results suggest that larotrectinib could potentially be used as a therapeutic agent aimed at mitigating NGF-mediated osteosarcoma progression.
Assuntos
Monócitos , Fator de Crescimento Neural , Osteossarcoma , Microambiente Tumoral , Molécula 1 de Adesão de Célula Vascular , Osteossarcoma/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Humanos , Fator de Crescimento Neural/metabolismo , Animais , Microambiente Tumoral/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismo , Camundongos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Macrófagos/metabolismo , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Camundongos NusRESUMO
PAK4 and PD-L1 have been suggested as novel therapeutic targets in human cancers. Moreover, PAK4 has been suggested to be a molecule closely related to the immune evasion of cancers. Therefore, this study evaluated the roles of PAK4 and PD-L1 in the progression of osteosarcomas in 32 osteosarcomas and osteosarcoma cells. In human osteosarcomas, immunohistochemical positivity for the expression of PAK4 (overall survival, p = 0.028) and PD-L1 (relapse-free survival, p = 0.002) were independent indicators for the survival of patients in a multivariate analysis. In osteosarcoma cells, the overexpression of PAK4 increased proliferation and invasiveness, while the knockdown of PAK4 suppressed proliferation and invasiveness. The expression of PAK4 was associated with the expression of the molecules related to cell cycle regulation, invasion, and apoptosis. PAK4 was involved in resistance to apoptosis under a treatment regime with doxorubicin for osteosarcoma. In U2OS cells, PAK4 was involved in the stabilization of PD-L1 from ubiquitin-mediated proteasomal degradation and the in vivo infiltration of immune cells such as regulatory T cells and PD1-, CD4-, and CD8-positive cells in mice tumors. In conclusion, this study suggests that PAK4 is involved in the progression of osteosarcoma by promoting proliferation, invasion, and resistance to doxorubicin and stabilized PD-L1 from proteasomal degradation.
Assuntos
Antígeno B7-H1 , Proliferação de Células , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Osteossarcoma , Quinases Ativadas por p21 , Osteossarcoma/patologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Osteossarcoma/genética , Humanos , Antígeno B7-H1/metabolismo , Feminino , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Animais , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Masculino , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Camundongos , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Adulto , Adolescente , Estabilidade Proteica/efeitos dos fármacos , Camundongos Nus , Adulto Jovem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Invasividade NeoplásicaRESUMO
Recent studies have highlighted the significant role of 5-hydroxymethylcytosine (5hmC) in carcinogenesis. However, the specific role of 5hmC in osteosarcoma (OS) remains largely unexplored. The-re-fore, this study aimed to investigate the function of 5hmC and TET3 in OS. In this study, we found a decreased total level of 5hmC in OS tissues. The expression of the TET3 protein was also decreased in OS. Importantly, the decreased levels of TET3 were associated with a decreased disease-free survival (DFS) rate in patients. To investigate the role of TET3 and 5hmC in OS, we manipulated the levels of TET3 in MG-63 cells. Silencing TET3 in these cells resulted in a twofold increase in proliferation. Additio-nally, the level of 5hmC decreased in these cells. Con-versely, over-expression of TET3 in MG-63 cells led to the expected inhibition of proliferation and invasion, accompanied by an increase in 5hmC levels. In conclusion, both 5hmC and TET3 protein levels were decreased in OS. Additionally, the over-expression of TET3 inhibited the proliferation of MG-63 cells, while the suppression of TET3 had the opposite effect. These findings suggest that decreased levels of 5hmC and TET3 may serve as potential markers for OS.
Assuntos
5-Metilcitosina , Proliferação de Células , Desmetilação do DNA , Dioxigenases , Epigênese Genética , Feminino , Humanos , Masculino , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Dioxigenases/metabolismo , Regulação Neoplásica da Expressão Gênica , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genéticaRESUMO
Osteosarcoma (OS) is the most frequent bone malignancy in humans. Previous evidence suggest that circ_0032463 is an oncogenic circular RNA (circRNA) in various cancers, including OS. However, the molecular mechanism of circ_0032463 involved in OS is still unclear. Circ_0032463, microRNA-145-5p (miR-145-5p), GDNF receptor alpha 1 (GFRA1), and Wilms tumor 1-associated protein (WTAP) levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, apoptosis, migration, invasion, and angiogenesis were analyzed using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and tube formation assays. Western blot analysis was performed to measure matrix metalloproteinase 2 (MMP2), MMP9, GFRA1, and WTAP protein levels. Binding between miR-145-5p and circ_0032463 or GFRA1 was confirmed using a dual-luciferase reporter and pull-down assay. The biological role of circ_0032463 on OS cell growth was also analyzed using a xenograft tumor model in vivo. Methylated RNA immunoprecipitation assay validated the interaction between WTAP and circ_0032463. Circ_0032463, GFRA1, and WTAP levels were increased, and miR-145-5p was decreased in OS tissues and cells. Circ_0032463 deficiency might hinder OS cell proliferation, migration, invasion, angiogenesis, and promote apoptosis in vitro. Mechanically, circ_0032463 worked as a miR-145-5p sponge to increase GFRA1 expression. Repression of circ_0032463 knockdown on tumor cell growth was proved in vivo. Besides, N6-methyladenosine (m6A) modification facilitates the biogenesis of circ_0032463. Taken together, m6A-mediated biogenesis of circ_0032463 facilitates OS cell malignant biological behavior partly via regulating the miR-145-5p/GFRA1 axis, suggesting a promising molecular marker for OS treatment.
Assuntos
Neoplasias Ósseas , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , MicroRNAs , Osteossarcoma , RNA Circular , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Regulação Neoplásica da Expressão Gênica , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Camundongos Nus , Masculino , Camundongos Endogâmicos BALB C , Proliferação de Células/genética , Progressão da Doença , Feminino , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Adenosina/análogos & derivados , Proteínas de Ciclo CelularRESUMO
As a subclass of noncoding RNAs, circular RNA play an important role in tumour development. The aim of this study was to investigate the role of circ_0004674 in osteosarcoma glycolysis and the molecular mechanism of its regulation. We examined the expression of circ_0004674, miR-140-3p, TCF4 and glycolysis-related proteins (including HK2, PKM2, GLUT1 and LDHA) in osteosarcoma cells and tissues by quantitative reverse transcription-polymerase chain reaction and immunoblotting (Western blot analysis). The role of circ_0004674, miR-140-3p and TCF4 in the proliferation, apoptosis, migration and invasion of OS cells was examined using CCK8 assay, Apoptosis assay, Wound healing assay, Transwell migration and Matrigel invasion assay. The interaction of circ_0004674/miR-140-3p and miR-1543/TCF4 was also analysed using a dual luciferase reporter assay. Finally, the glycolytic process was assessed by glucose uptake assays and lactate production measurements. The results showed that the expression of circ_0004674 and TCF4 was significantly higher in MG63 and U2OS cells compared to hFOB1.19 cells, while the expression of miR-140-3p was downregulated. Silencing of circ_0004674 gene significantly inhibited the proliferation, migration and invasion of cancer cells and promoted apoptosis of cancer cells. Experiments such as dual luciferase reporter analysis showed that circ_0004674 regulates the expression of glycolysis-related proteins through the miR-140-3p/TCF4 pathway, and inhibition of this gene attenuated the depletion of glucose content and the production of lactate in cancer cells. Furthermore, inhibition of miR-140-3p or overexpression of TCF could reverse the phenotypic changes in cancer cells induced by circ_0004674 silencing. In summary, this study elucidated the specific function and potential mechanisms of circ_0004674 in osteosarcoma glycolysis. The findings demonstrate that miR-140-3p and TCF4 function respectively as a tumor suppressor gene and an oncogene in osteosarcoma. Notably, they influence glycolysis and associated pathways, regulating osteosarcoma proliferation. Therefore, circ_0004674 promotes osteosarcoma glycolysis and proliferation through the miR-140-3p/TCF4 pathway, enhancing the malignant behaviour of tumours, and it is expected to be a potential molecular target for osteosarcoma treatment.
Assuntos
Proliferação de Células , Glicólise , MicroRNAs , Osteossarcoma , RNA Circular , Fator de Transcrição 4 , Humanos , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Linhagem Celular Tumoral , RNA Circular/genética , RNA Circular/metabolismo , Fator de Transcrição 4/metabolismo , Fator de Transcrição 4/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Apoptose/genética , Transdução de SinaisRESUMO
Metastasis is the leading cause of cancerrelated death in osteosarcoma (OS). OS stem cells (OSCs) and anoikis resistance are considered to be essential for tumor metastasis formation. However, the underlying mechanisms involved in the maintenance of a stemcell phenotype and anoikis resistance in OS are mostly unknown. Foslike antigen 1 (FOSL1) is important in maintaining a stemlike phenotype in various cancers; however, its role in OSCs and anoikis resistance remains unclear. In the present study, the dynamic expression patterns of FOSL1 were investigated during the acquisition of cancer stemlike properties using RNA sequencing, PCR, western blotting and immunofluorescence. Flow cytometry, tumorsphere formation, clone formation assays, anoikis assays, western blotting and in vivo xenograft and metastasis models were used to further investigate the responses of the stemcell phenotype and anoikis resistance to FOSL1 overexpression or silencing in OS cell lines. The underlying molecular mechanisms were evaluated, focusing on whether SOX2 is crucially involved in FOSL1mediated stemness and anoikis in OS. FOSL1 expression was observed to be upregulated in OSCs and promoted tumorsphere formation, clone formation and tumorigenesis in OS cells. FOSL1 expression correlated positively with the expression of stemnessrelated factors (SOX2, NANOG, CD117 and Stro1). Moreover, FOSL1 facilitated OS cell anoikis resistance and promoted metastases by regulating the expression of apoptosis related proteins BCL2 and BAX. Mechanistically, FOSL1 upregulated SOX2 expression by interacting with the SOX2 promoter and activating its transcription. The results also showed that SOX2 is critical for FOSL1mediated stemlike properties and anoikis resistance. The current findings indicated that FOSL1 is an important regulator that promotes a stem celllike phenotype and anoikis resistance to facilitate tumorigenesis and metastasis in OS by regulating the transcription of SOX2. Thus, FOSL1 might represent an attractive target for therapeutic interventions in OS.
Assuntos
Anoikis , Carcinogênese , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas , Osteossarcoma , Proteínas Proto-Oncogênicas c-fos , Fatores de Transcrição SOXB1 , Osteossarcoma/patologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Anoikis/genética , Animais , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Camundongos , Carcinogênese/genética , Carcinogênese/patologia , Metástase Neoplásica , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Camundongos Nus , Masculino , Feminino , Camundongos Endogâmicos BALB CRESUMO
OBJECTIVES: Osteosarcoma is the most common malignant bone tumor in children and adolescents, characterized by a high potential for proliferation and metastasis. Patients with osteosarcoma who have distant metastases generally have a poor prognosis. Challenges in treatment include incomplete resection of tumor and chemotherapy resistance, with no effective cure currently available. Recent studies suggest that ß-1,4-N-acetyl-galactosaminyltransferase 1 (B4GALNT1) plays a role in the progression of various malignant tumors. However, the function of B4GALNT1 in osteosarcoma cells has not been reported. This study aims to investigate the expression of B4GALNT1 in osteosarcoma tissues compared to normal tissues and to explore its effects on the proliferation, migration, and invasion of osteosarcoma cells, thereby providing new theoretical foundations and directions for the treatment of osteosarcoma patients. METHODS: Tumor tissues and corresponding normal tissue samples were collected from 16 osteosarcoma patients who underwent tumor resection at the Second Xiangya Hospital of Central South University. The patients' ages ranged from 8 to 17 years (median age 12 years). The expression of B4GALNT1 mRNA in osteosarcoma tissues, corresponding normal tissues, 3 osteosarcoma cell lines (MG63, Saos-2, and U2OS), and human fetal osteoblastic cells (hFOB) was detected using real-time reverse transcription PCR (real-time RT-PCR). The effects of B4GALNT1 knockdown on the proliferation of osteosarcoma cells Saos-2 and U2OS were analyzed using cell counting kit-8 (CCK-8) assays and colony formation assays. The effects of B4GALNT1 knockdown on the migration and invasion abilities of Saos-2 and U2OS cells were evaluated using Transwell migration and invasion assays. Western blotting analysis was performed to assess the impact of B4GALNT1 knockdown on the expression of epithelial-mesenchymal transition (EMT) and invasion-related proteins in Saos-2 and U2OS cells. RESULTS: Real-time RT-PCR results showed that B4GALNT1 mRNA expression levels were significantly higher in osteosarcoma tissues and the 3 osteosarcoma cell lines compared to normal tissues and hFOB cells (all P<0.01). CCK-8 and colony formation assays indicated that B4GALNT1 knockdown significantly reduced the proliferation rate of osteosarcoma cells compared to the control group (all P<0.05). Transwell migration and invasion assays demonstrated that B4GALNT1 knockdown significantly decreased the number of migrating and invading osteosarcoma cells (all P<0.01). Western blotting analysis revealed that B4GALNT1 knockdown inhibited the expression of N-cadherin, Snail, Vimentin, and matrix metalloproteinase 9 (MMP9) compared to the control group (all P<0.01). CONCLUSIONS: B4GALNT1 is upregulated in osteosarcoma tissues and cell lines, and its knockdown suppresses the malignant phenotype of osteosarcoma cells. B4GALNT1 may function as an oncogene in the proliferation and metastasis of osteosarcoma cells.
Assuntos
Neoplasias Ósseas , Movimento Celular , Proliferação de Células , Regulação para Baixo , N-Acetilgalactosaminiltransferases , Osteossarcoma , Humanos , Osteossarcoma/patologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Proliferação de Células/genética , Criança , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Adolescente , Linhagem Celular Tumoral , Movimento Celular/genética , Masculino , Feminino , Invasividade Neoplásica , Polipeptídeo N-Acetilgalactosaminiltransferase , Metástase Neoplásica , RNA Interferente Pequeno/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Corilagin (CLG) has antitumor activities in certain human malignant cancers. Herein, the effects and mechanisms of CLG on osteosarcoma (OS) were investigated. OS cell viability and proliferation were detected by MTT and colony formation assay. Cell cycle and apoptosis were examined using flow cytometry. The interaction between TRAF6 and FLT3 was investigated using a co-immunoprecipitation assay. Results demonstrated that CLG treatment inhibited OS cell viability and proliferation but promoted OS cell autophagy and apoptosis in a concentration-dependent manner. Mechanically, CLG inhibited TRAF6-mediated FLT3 ubiquitination degradation. TRAF6 overexpression abolished the effects of CLG on OS cell proliferation, autophagy, and apoptosis. Finally, CLG administration inhibited OS tumor growth in mice by inducing autophagy-dependent apoptosis. Taken together, CLG inhibited OS progression by facilitating mTOR/ULK1 pathway-mediated autophagy through inhibiting TRAF6-mediated FLT3 ubiquitination, which indicated that CLG was a promising candidate for the treatment of OS.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Proliferação de Células , Osteossarcoma , Fator 6 Associado a Receptor de TNF , Serina-Treonina Quinases TOR , Ubiquitinação , Tirosina Quinase 3 Semelhante a fms , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/efeitos dos fármacos , Humanos , Animais , Camundongos , Serina-Treonina Quinases TOR/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Sobrevivência Celular/efeitos dos fármacosRESUMO
Osteosarcoma is a highly metastatic, aggressive bone cancer that occurs in children and young adults worldwide. Circular RNAs (circRNAs) are crucial molecules for osteosarcoma progression. In this study, we aimed to investigate the impact of circMRPS35 overexpression and its interaction with FOXO1 via evaluating apoptosis, cell cycle, and bioinformatic analyses on the malignant development of osteosarcoma in MG63 and MNNG/HOS cells. We found that circMRPS35 overexpression reduced osteosarcoma cell viability and inhibited tumor growth in vivo. It increased the apoptosis rate and induced cell cycle arrest in osteosarcoma cells. We identified a potential interaction between circMRPS35 and FOXO1 with miR-105-5p using bioinformatics analysis. Overexpression of circMRPS35 decreased miR-105-5p expression, whereas miR-105-5p mimic treatment increased its expression. This mimic also suppressed the luciferase activity of circMRPS35 and FOXO1 and reduced FOXO1 expression. Overexpression of circMRPS35 elevated FOXO1 protein levels, but this effect was reversed by co-treatment with the miR-105-5p mimic. We demonstrated that inhibiting miR-105-5p decreased viability and induced apoptosis. Overexpression of FOXO1 or treatment with a miR-105-5p inhibitor could counteract the effects of circMRPS35 on viability and apoptosis in osteosarcoma cells. Therefore, we concluded that circMRPS35 suppressed the malignant progression of osteosarcoma via targeting the miR-105-5p/FOXO1 axis.
Assuntos
Apoptose , Neoplasias Ósseas , Proteína Forkhead Box O1 , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Osteossarcoma , RNA Circular , Animais , Humanos , Camundongos , Apoptose/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Progressão da Doença , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , RNA Circular/genética , RNA Circular/metabolismoRESUMO
ARHGAP25, a member of the ARHGAP family, encodes a negative regulator of Rho-GTPase that is important for actin remodeling, cell polarity, and cell migration. ARHGAP25 is down-regulated in a variety of solid tumors and promotes cancer cell growth, migration, and invasion. However, nothing is understood about ARHGAP25's biological function in osteosarcoma. This work used qPCR and WB to confirm the expression of ARHGAP25 in osteosarcoma following the initial analysis of its expression in pan-cancer. For GO and KEGG analysis, we have chosen 300 genes from the TARGET osteosarcoma data that had the strongest positive correlation with ARHGAP25, and we created nomogram and calibration charts. We simultaneously overexpressed ARHGAP25 in osteosarcoma cells to examine its impact on apoptosis and proliferation. By using MSP, we determined their methylation status in osteosarcoma cells and normal bone cells. We observed that ARHGAP25 was significantly downregulated in a range of malignancies, including osteosarcoma, and was associated with poor patient outcomes. The decrease of ARHGAP25 expression in osteosarcoma is related to DNA methylation. Overexpression of ARHGAP25 induced apoptosis and inhibited the proliferation of osteosarcoma cells in vitro. In addition, ARHGAP25 is also associated with immune-related pathways in osteosarcoma. These findings suggest that ARHGAP25 is a valuable prognostic biomarker in osteosarcoma patients.
Assuntos
Apoptose , Neoplasias Ósseas , Proliferação de Células , Biologia Computacional , Metilação de DNA , Proteínas Ativadoras de GTPase , Regulação Neoplásica da Expressão Gênica , Osteossarcoma , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Humanos , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Biologia Computacional/métodos , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/mortalidade , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Prognóstico , Masculino , Feminino , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Relevância ClínicaRESUMO
BACKGROUND: Ring finger protein 135 (RNF135) is an E3 ubiquitin ligase that has been implicated in the tumorigenesis of multiple human malignancies. However, whether RNF135 plays a role in the development of human osteosarcoma (OS) remains unknown. METHODS: RNF135 expression in 20 human OS and 20 human osteochondroma specimens were evaluated by means of immunohistochemistry staining. The effects of shRNA-mediated RNF135 knockdown on human OS cell growth and apoptosis were evaluated through a panel of in vitro studies on cell proliferation, colony formation, exposure of phosphatidylserine on the cell surface, and caspase 3/7 activation. The protein levels of PI3K, AKT, and p-AKT were determined by western blot analysis. RESULTS: We detected significantly higher RNF135 levels in human OS tissues than human osteochondroma tissues. In in vitro studies, shRNA-mediated RNF135 knockdown in human OS cells inhibited proliferation and induced apoptosis. In addition, RNF135 knockdown reduced PI3K and p-AKT protein levels and activated caspase 3 and 7. CONCLUSIONS: These results supported that RNF135 contributes to human OS development through PI3K/AKT-dependent mechanisms. Targeting RNF135 may provide a new therapeutic approach for treating this human malignancy.
Assuntos
Apoptose , Neoplasias Ósseas , Proliferação de Células , Osteossarcoma , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Ubiquitina-Proteína Ligases , Feminino , Humanos , Masculino , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Osteocondroma/patologia , Osteocondroma/genética , Osteocondroma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Osteosarcoma is a soft tissue neoplasm with elevated recurrence risk and highly metastatic potential. Metal response element binding transcriptional factor 2 (MTF2) has been revealed to exert multiple activities in human tissues. The present research was conducted to explore the functions and related response mechanism of MTF2 in osteosarcoma which have not been introduced yet. METHODS: Bioinformatics tools identified the differential MTF2 expression in osteosarcoma tissues. MTF2 expression in osteosarcoma cells was examined with Western blot. Cell Counting Kit-8 (CCK-8) assay, 5-Ethynyl-2'-deoxyuridine (EDU) staining, wound healing as well as transwell assays measured cell proliferation, migration and invasion, respectively. Flow cytometry assay detected the cellular apoptotic level. Western blot also measured the expressions of proteins associated with epithelial mesenchymal transition (EMT), apoptosis and enhancer of zeste homolog 2 (EZH2)/secreted frizzled-related protein 1 (SFRP1)/Wnt signaling. Co-immunoprecipitation (Co-IP) assay confirmed MTF2-EZH2 interaction. RESULTS: MTF2 expression was increased in osteosarcoma tissues and cells. MTF2 interference effectively inhibited the proliferation, migration and invasion of osteosarcoma cells and promoted the cellular apoptotic rate. MTF2 directly bound to EZH2 and MTF2 silence reduced EZH2 expression, activated SFRP1 expression and blocked Wnt signaling in osteosarcoma cells. EZH2 upregulation or SFRP1 antagonist WAY-316606 partly counteracted the impacts of MTF2 down-regulation on the SFRP1/Wnt signaling and the biological phenotypes of osteosarcoma cells. CONCLUSIONS: MTF2 might down-regulate SFRP1 to activate Wnt signaling and drive the progression of osteosarcoma via interaction with EZH2 protein.
Assuntos
Neoplasias Ósseas , Proliferação de Células , Proteína Potenciadora do Homólogo 2 de Zeste , Osteossarcoma , Via de Sinalização Wnt , Humanos , Apoptose/fisiologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Via de Sinalização Wnt/fisiologia , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismoRESUMO
WNT signaling regulates osteosarcoma proliferation. However, there is controversy in the field of osteosarcoma as to whether WNT signaling is pro- or anti-tumorigenic. WNT-targeting therapeutics, both activators and inhibitors, are compared. WNT5B, a ß-catenin-independent ligand, and WNT10B, a ß-catenin-dependent WNT ligand, are each expressed in osteosarcomas, but they are not expressed in the same tumors. Furthermore, WNT10B and WNT5B regulate different histological subtypes of osteosarcomas. Using WNT signaling modulators as therapeutics may depend on the WNT ligand and/or the activated signaling pathway.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Proteínas Wnt , Via de Sinalização Wnt , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/tratamento farmacológico , Humanos , Proteínas Wnt/metabolismo , Proteínas Wnt/antagonistas & inibidores , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Terapia de Alvo Molecular , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , beta Catenina/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
BACKGROUND: Osteosarcoma (OS) is one of the most common primary malignant tumors of bone in children, which develops from osteoblasts and typically occurs during the rapid growth phase of the bone. Recently, Super-Enhancers(SEs)have been reported to play a crucial role in osteosarcoma growth and metastasis. Therefore, there is an urgent need to identify specific targeted inhibitors of SEs to assist clinical therapy. This study aimed to elucidate the role of BRD4 inhibitor GNE-987 targeting SEs in OS and preliminarily explore its mechanism. METHODS: We evaluated changes in osteosarcoma cells following treatment with a BRD4 inhibitor GNE-987. We assessed the anti-tumor effect of GNE-987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, xenograft tumor size measurements, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism. RESULTS: In this study, we found that extremely low concentrations of GNE-987(2-10 nM) significantly reduced the proliferation and survival of OS cells by degrading BRD4. In addition, we found that GNE-987 markedly induced cell cycle arrest and apoptosis in OS cells. Further study indicated that VHL was critical for GNE-987 to exert its antitumor effect in OS cells. Consistent with in vitro results, GNE-987 administration significantly reduced tumor size in xenograft models with minimal toxicity, and partially degraded the BRD4 protein. KRT80 was identified through analysis of the RNA-seq and ChIP-seq data. U2OS HiC analysis suggested a higher frequency of chromatin interactions near the KRT80 binding site. The enrichment of H3K27ac modification at KRT80 was significantly reduced after GNE-987 treatment. KRT80 was identified as playing an important role in OS occurrence and development. CONCLUSIONS: This research revealed that GNE-987 selectively degraded BRD4 and disrupted the transcriptional regulation of oncogenes in OS. GNE-987 has the potential to affect KRT80 against OS.