RESUMO
Retinoblastoma (RB) is the most common intraocular malignancy among children and presents a certain mortality risk, especially in low- and middle-income countries. Clarifying the molecular mechanisms underlying the onset and progression of retinoblastoma is vital for devising effective cancer treatment approaches. PRMT1, a major type I PRMT, plays significant roles in cancer development. However, its expression and role in retinoblastoma are still unclear. Our research revealed a marked increase in PRMT1 levels in both retinoblastoma tissues and Y79 cells. The overexpression of PRMT1 in Y79 cells promoted their growth and cell cycle progression. Conversely, the suppression of PRMT1 hindered the growth of Y79 cells and impeded cell cycle progression. Mechanistically, PRMT1 mediated the growth of Y79 retinoblastoma cells by targeting the p53/p21/CDC2/Cyclin B pathway. Additionally, the ability of PRMT1 knockdown to suppress cell proliferation was also observed in vivo. Overall, PRMT1 could function as a potential target for therapeutic treatment in individuals with retinoblastoma.
Assuntos
Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21 , Proteína-Arginina N-Metiltransferases , Proteínas Repressoras , Neoplasias da Retina , Retinoblastoma , Proteína Supressora de Tumor p53 , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Retinoblastoma/patologia , Retinoblastoma/metabolismo , Retinoblastoma/genética , Humanos , Proliferação de Células/fisiologia , Neoplasias da Retina/patologia , Neoplasias da Retina/metabolismo , Neoplasias da Retina/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC2/genética , Regulação Neoplásica da Expressão Gênica , Animais , Camundongos , Western Blotting , Ciclo Celular/fisiologia , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Linhagem Celular Tumoral , Camundongos NusRESUMO
PURPOSE: Retinoblastoma (RB) is one of the most common intraocular cancers, with the highest prevalence among infants and young children under the age five. Numerous findings across the literature illustrate the involvement and significance of circular RNAs (circRNAs) in human malignancies, including RB. The current investigation attempted to decipher the exact roles and underlying mechanisms of a novel circRNA, hsa_circ_0078136, in RB progression. METHODS: The hsa_circ_0078136 expression was evaluated in RB tumors and cell lines via qRT-PCR. The significance of hsa_circ_0078136 in RB was examined by performing CCK8 assay, transwell assays, western blotting of apoptotic and IL-17 signaling ligand molecules, and a subcutaneous xenograft tumor model. In addition, the interaction of circRNA and eukaryotic translation initiation factor 4A3 (EIF4A3) was determined with bioinformatics, western blot, and RIP assay. RESULTS: The hsa_circ_0078136 expression was reduced in RB tumor samples and cells. Additionally, its overexpression restricted the oncogenic properties of RB cells in vitro. Moreover, hsa_circ_0078136 overexpression lowered the protein levels of cytokine ligand molecules of IL-17 signaling pathway in RB cell lines. In vivo, hsa_circ_0078136 overexpression in subcutaneous tumor xenografts reduced tumor growth. We also observed that EIF4A3 binds to the downstream flanking sequence of hsa_circ_0078136 in the SHRPH pre-mRNA transcript, and EIF4A3 overexpression reduced hsa_circ_0078136 expression, suggesting that EIF4A3 inhibited hsa_circ_0078136 formation. CONCLUSIONS: Our results demonstrate that hsa_circ_0078136 is regulated by EIF4A3 and functions as a tumor suppressor via the IL-17 signaling pathway in RB.
Assuntos
Carcinogênese , Fator de Iniciação 4A em Eucariotos , Interleucina-17 , RNA Circular , Neoplasias da Retina , Retinoblastoma , Transdução de Sinais , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Humanos , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologia , RNA Circular/genética , Interleucina-17/metabolismo , Interleucina-17/genética , Camundongos , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Animais , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Camundongos Nus , Apoptose , Masculino , Células Tumorais Cultivadas , Linhagem Celular Tumoral , Feminino , RNA Helicases DEAD-boxRESUMO
Retinoblastoma is the most common pediatric intraocular malignant tumor affecting 1:15 000-1:20 000 live births. Even though the survival rate in developed countries is over 90â¯%, more efficient treatment options are needed for better vision salvage and reduction of the adverse effects. Therefore, we investigated fluorescein-labeled PL3 peptide targeting properties towards the Y79 retinoblastoma cell line in vitro. Through the application of cellular imaging and flow cytometry techniques, the PL3 peptide exhibited a rapid and specific internalization within Y79 cells, with subsequent translocation to the cell nuclei, showcasing notable accumulation in the nucleoli. This phenomenon was not present in other investigated cell lines and was not observable with similarly charged and length control peptide. However, the exact mechanism behind this Y79 cell line-specific nuclear and nucleolar targeting pattern remains elusive. In the future, this targeting process could facilitate specific treatment modalities of retinoblastoma with PL3 peptide-coupled drug delivery technologies.
Assuntos
Nucléolo Celular , Retinoblastoma , Retinoblastoma/metabolismo , Retinoblastoma/tratamento farmacológico , Humanos , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Nucléolo Celular/efeitos dos fármacos , Peptídeos/farmacologia , Neoplasias da Retina/metabolismo , Neoplasias da Retina/tratamento farmacológico , Peptídeos Penetradores de Células/farmacologiaRESUMO
Retinoblastoma (RB) is a pediatric malignancy, typically diagnosed at birth or during early childhood. The pathogenesis of RB is marked by the amplification of the Basic Helix-Loop-Helix (BHLH) Transcription Factor MYCN, which serves as a transcriptional regulator capable of binding to Dickkopf 3 (DKK3). However, the precise role of DKK3 in the malignant progression of RB cells caused by MYCN remains elusive. In the present study, the expression of MYCN was either overexpressed or interfered in RB cells. Subsequently, the expression level of DKK3 was assessed through quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation was evaluated using the Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine staining, while cell cycle progression and apoptosis were analyzed by flow cytometry and western blot analysis, respectively. Additionally, the expression of proteins involved in the Wnt/ß-catenin/Fra-1/p53 signaling pathway was evaluated via western blot analysis. To gain further insights, Wnt agonists and the P53 inhibitor PFT-α were introduced into exploration. The current investigation revealed a negative correlation between the expression levels of MYCN and DKK3 in RB cells. Additionally, DKK3 overexpression inhibited cell proliferation, promoted cell apoptosis, and arrested cell cycle in RB cells with high expression of MYCN. Moreover, enhanced DKK3 expression inhibited proliferation, promoted cell cycle arrest and apoptosis of RB cells by modulating the wnt/ßcatenin/Fra-1/p53 signaling pathway. Furthermore, in vivo experiments revealed that overexpression of DKK3 inhibits the growth of RB tumors. Collectively, our findings elucidate that MYCN stimulates the Wnt/ß-catenin/Fra-1 pathway by suppressing DKK3 expression, ultimately suppressing p53 activity and contributing to malignant progression of RB.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proliferação de Células , Proteína Proto-Oncogênica N-Myc , Retinoblastoma , Proteína Supressora de Tumor p53 , Via de Sinalização Wnt , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Retinoblastoma/metabolismo , Retinoblastoma/genética , Retinoblastoma/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Animais , Camundongos , Apoptose , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos Nus , beta Catenina/metabolismoRESUMO
Retinoblastoma, a pediatric ocular malignancy, presents significant challenges in comprehending its molecular underpinnings and targeted therapeutic approaches. The dysregulated activity of histone deacetylases (HDACs) has been associated with retinoblastoma pathogenesis, influencing critical cellular processes like cell cycle regulation or retinal ganglion cell apoptosis. Through their deacetylase activity, HDACs exert control over key tumor suppressors and oncogenes, influencing the delicate equilibrium between proliferation and cell death. Furthermore, the interplay between HDACs and the retinoblastoma protein pathway, a pivotal aspect of retinoblastoma etiology, reveals a complex network of interactions influencing the tumor microenvironment. The examination of HDAC inhibitors, encompassing both established and novel compounds, offers insights into potential approaches to restore acetylation balance and impede retinoblastoma progression. Moreover, the identification of specific HDAC isoforms exhibiting varying expression in retinoblastoma provides avenues for personalized therapeutic strategies, allowing for interventions tailored to individual patient profiles. This review focuses on the intricate interrelationship between HDACs and retinoblastoma, shedding light on epigenetic mechanisms that control tumor development and progression. The exploration of HDAC-targeted therapies underscores the potential for innovative treatment modalities in the pursuit of more efficacious and personalized management strategies for this disease.
Assuntos
Inibidores de Histona Desacetilases , Histona Desacetilases , Retinoblastoma , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Humanos , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Inibidores de Histona Desacetilases/uso terapêutico , Inibidores de Histona Desacetilases/farmacologia , Animais , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologia , Epigênese Genética , Acetilação , Microambiente Tumoral , Regulação Neoplásica da Expressão Gênica , Proteína do Retinoblastoma/metabolismo , Proteína do Retinoblastoma/genéticaRESUMO
Retinoblastoma (RB) is an intraocular malignancy initiated by loss of RB1 function and/or dysregulation of MYCN oncogene. RB is primarily treated with chemotherapy; however, systemic toxicity and long-term adverse effects remain a significant challenge necessitating the identification of specific molecular targets. Aurora kinase A (AURKA), a critical cell cycle regulator, contributes to cancer pathogenesis, especially in RB1-deficient and MYCN-dysregulated tumors. The current immunohistochemistry study in patient specimens (n = 67) indicated that AURKA is overexpressed in RB, and this elevated expression correlates with one or more histopathologic high-risk factors, such as tumor involvement of the optic nerve, choroid, sclera, and/or anterior segment. More specifically, AURKA is ubiquitously expressed in most advanced-stage RB tumors that show a suboptimal response to chemotherapy. shRNA-mediated depletion/pharmacologic inhibition studies in cell lines, patient-derived cells, in vivo xenografts, and enucleated patient specimens confirmed that RB cells are highly sensitive to a lack of functional AURKA. In addition, AURKA and N-myc proto-oncogene protein (MYCN) associate with each other to regulate their levels in RB cells. Overall, these results demonstrate a previously unknown up-regulation of AURKA in RB, facilitated by its crosstalk with MYCN. The elevated levels of this kinase may indicate unfavorable prognosis in tumors refractory to chemotherapy. This study provides a rationale and confirms that therapeutic targeting of elevated AURKA in RB could be a potential treatment approach.
Assuntos
Aurora Quinase A , Proto-Oncogene Mas , Neoplasias da Retina , Retinoblastoma , Pré-Escolar , Feminino , Humanos , Masculino , Aurora Quinase A/metabolismo , Aurora Quinase A/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Terapia de Alvo Molecular , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Neoplasias da Retina/patologia , Neoplasias da Retina/metabolismo , Neoplasias da Retina/genética , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/patologia , Retinoblastoma/metabolismo , Retinoblastoma/genética , Fatores de Risco , Animais , Embrião de GalinhaRESUMO
In this study, we tested the hypothesis that ALYREF/THOC4, a poor prognostic factor in different cancer types, has potential as a drug target and prognostic biomarker for retinoblastoma (RB). Immunostaining (IHC), Western blot, and RT-qPCR analyses detected overexpression of ALYREF in the RB cell lines Y79, RB143, WERI-RB1, and RB116. IHC analysis on RB tumor array showed that 11/14 of RB tumors were ALYREF+ to varying degrees, with eight tumors at maximum 3+ intensity. The IHC analysis also detected ALYREF+ cells in normal retina, mainly in the inner nuclear and ganglion cell layer, while some tumor-bearing human eyes were ALYREF+ in the optic nerve suggesting a role in optic invasion/tumor invasion. The expression of ALYREF within the tumor itself, in the optic nerve, as well as in adjacent "normal" retina, suggest that this pattern of expression may lead to ALYREF being a potentially useful prognostic indicator for RB, as it is for other tumors. siRNA knockdown of ALYREF resulted in a 40â¯% decrease in cell growth in both WERI-RB1 and Y79 cells (p<0.05) and this was associated with decreased expression of mRNAs for the cell proliferation markers Ki67 and PCNA (p<0.005). These results suggest a role for ALYREF in RB cell growth regulation and its potential as both a target and a biomarker for tumor growth inhibition by anti-cancer therapies.
Assuntos
Proliferação de Células , Neoplasias da Retina , Retinoblastoma , Retinoblastoma/patologia , Retinoblastoma/metabolismo , Retinoblastoma/genética , Humanos , Neoplasias da Retina/patologia , Neoplasias da Retina/metabolismo , Neoplasias da Retina/genética , Linhagem Celular Tumoral , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genéticaRESUMO
Retinoblastoma (RB) is a pediatric cancer of the eye that occurs in 1/15000 live births worldwide. Albeit RB is initiated by the inactivation of RB1 gene, the disease progression relies largely on transcriptional alterations. Therefore, evaluating gene expression is vital to unveil the therapeutic targets in RB management. In this study, we employed an RT2 Profiler™ PCR array for a focused analysis of 84 cancer-specific genes in RB. An interaction network was built with gene expression data to identify the dysregulated pathways in RB. The key transcript alterations identified in 13 tumors by RT2 Profiler™ PCR array was further validated in 15 tumors by independent RT-qPCR. Out of 84 cancer-specific genes, 68 were dysregulated in RB tumors. Among the 68 genes, 23 were chosen for further analysis based on statistical significance and abundance across multiple tumors. Pathway analysis of altered genes showed the frequent perturbations of cell cycle, angiogenesis and apoptotic pathways in RB. Notably, upregulation of MCM2, MKI67, PGF, WEE1, CDC20 and downregulation of COX5A were found in all the tumors. Western blot confirmed the dysregulation of identified targets at protein levels as well. These alterations were more prominent in invasive RB, correlating with the disease pathogenesis. Our molecular analysis thus identified the potential therapeutic targets for improving retinoblastoma treatment. We also suggest that PCR array can be used as a tool for rapid and cost-effective gene expression analysis.
Assuntos
Neoplasias da Retina , Retinoblastoma , Retinoblastoma/genética , Retinoblastoma/patologia , Retinoblastoma/metabolismo , Humanos , Neoplasias da Retina/genética , Neoplasias da Retina/patologia , Neoplasias da Retina/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão GênicaRESUMO
Purpose: Regression of retinoblastoma vitreous seeds (VS) during intravitreal chemotherapy can be delayed, resulting in supernumerary injections. Similarly, VS relapse may not be clinically evident at first. A predictive biomarker of tumor regression and relapse could help guide real-time clinical decision making. Retinoblastoma is an oxygen-sensitive tumor; paradoxically, VS survive in the hypoxic vitreous. We hypothesized that VS elaborate pro-angiogenic cytokines. The purpose was to determine if pro-angiogenic cytokine signatures from aqueous humor could serve as a biomarker of VS response to treatment. Methods: Multiplex ELISA was performed on aqueous from rabbit eyes with human retinoblastoma VS xenografts to identify expressed proangiogenic cytokines and changes in aqueous cytokine levels during intravitreal treatment were determined. Confirmatory RNAscope in situ hybridization for VEGF-A was performed on human retinoblastoma tumor sections and VS xenografts from rabbits. For human eyes undergoing intravitreal chemotherapy, serial aqueous VEGF-A levels measured via VEGF-A-specific ELISA were compared to clinical response. Results: VEGF-A was highly expressed in human retinoblastoma VS in the xenograft model, and was the only proangiogenic cytokine that correlated with VS disease burden. In rabbits, aqueous VEGF-A levels decreased in response to therapy, consistent with quantitative VS reduction. In patients, aqueous VEGF-A levels associated with clinical changes in disease burden (regression, stability, or relapse), with changes in VEGF-A levels correlating with clinical response. Conclusions: Aqueous VEGF-A levels correlate with extent of retinoblastoma VS, suggesting that aqueous VEGF-A may serve as a predictive molecular biomarker of treatment response.
Assuntos
Humor Aquoso , Biomarcadores Tumorais , Ensaio de Imunoadsorção Enzimática , Injeções Intravítreas , Neoplasias da Retina , Retinoblastoma , Fator A de Crescimento do Endotélio Vascular , Corpo Vítreo , Retinoblastoma/metabolismo , Retinoblastoma/tratamento farmacológico , Retinoblastoma/patologia , Animais , Neoplasias da Retina/metabolismo , Neoplasias da Retina/tratamento farmacológico , Neoplasias da Retina/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Humor Aquoso/metabolismo , Humanos , Corpo Vítreo/metabolismo , Corpo Vítreo/patologia , Coelhos , Biomarcadores Tumorais/metabolismo , Biópsia Líquida/métodos , Inoculação de Neoplasia , Feminino , Inibidores da Angiogênese/uso terapêutico , Citocinas/metabolismoRESUMO
Retinoblastoma is one of the most common ocular malignancies in children. Bmi-1, a member of the Polycomb group family of transcriptional repressors, is expressed in a variety of tumors. The purpose of our study was to explore the role of Bmi-1 in retinoblastoma. RT-qPCR and western blot were used for calculating the mRNA and protein levels of Bmi-1 and RKIP. MTT, Wound healing and Transwell assays were performed to measure the proliferation, migration and invasion in retinoblastoma cells. Cell apoptosis was detected by flow cytometry. The volume and mass of transplanted tumors were detected in nude mice. Bmi-1 was over expressed, and RKIP was low expressed in retinoblastoma cells. Bmi-1 promoted cell proliferation, migration and invasion and suppressed cell apoptosis of Y79 and SO-RB50 cells. Downregulation of Bmi-1 and overexpression of RKIP inhibited cell proliferation, migration and invasion, and increased cell apoptosis. The functions of Bmi-1 knockdown on retinoblastoma cells were blocked by RKIP knockdown, but promoted by RKIP. Down-regulated Bmi-1 inhibited xenograft tumor growth, and RKIP exacerbated this inhibitory effect. Bmi-1 served as a potential therapeutic target for improving the efficacy of clinical treatment in retinoblastoma. All the findings revealed the functions of Bmi-1/RKIP axis in retinoblastoma tumorigenesis.
Assuntos
Apoptose , Movimento Celular , Proliferação de Células , Camundongos Nus , Invasividade Neoplásica , Proteína de Ligação a Fosfatidiletanolamina , Complexo Repressor Polycomb 1 , Retinoblastoma , Humanos , Retinoblastoma/patologia , Retinoblastoma/metabolismo , Retinoblastoma/genética , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/genética , Apoptose/genética , Movimento Celular/genética , Animais , Linhagem Celular Tumoral , Camundongos , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias da Retina/patologia , Neoplasias da Retina/metabolismo , Neoplasias da Retina/genéticaRESUMO
Chromosomal abnormalities that involve the MYCN gene are rare; however, it is one of the most commonly mutated genes in retinoblastoma (RB) after the RB1 gene. MYCN is amplified in approximately 1-9 % of all RB tumors. It plays a role in RB oncogenesis via many mechanisms, including synergism with RB1 deletion, positive feedback with MDM2, upregulation of cell cycle regulating genes, upregulation of miRNA, and upregulation of glucose metabolism. MYCN amplifications are not mutually exclusive and can occur even in the presence of RB1 gene mutations. Clinically, RB1+/+MYCNA tumors present as sporadic, unilateral, advanced tumors in very young children and tend to follow an aggressive course. Magnetic resonance imaging features include peripheral tumor location, placoid configuration, retinal folding, tumor-associated hemorrhage, and anterior chamber enhancement. Genetic testing for MYCNA is especially recommended in patients with unilateral RB where genetic blood testing and tumor tissue show a lack of RB1 mutation. MYCN-targeted therapies are evolving and hold promise for the future.
Assuntos
Proteína Proto-Oncogênica N-Myc , Neoplasias da Retina , Retinoblastoma , Humanos , Retinoblastoma/genética , Retinoblastoma/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Neoplasias da Retina/diagnóstico , Mutação , Proteínas de Ligação a Retinoblastoma/genética , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: Recent in vitro studies using RB1+/- fibroblasts and MSCs have shown molecular and functional disruptions without the need for biallelic loss of RB1. However, this was not reflected in the recent in vitro studies employing RB1+/- retinal organoids. To gain further insights into the molecular disruptions in the RB1+/- retinal organoids, we performed a high throughput RNA sequencing analysis. METHODS AND RESULTS: iPSCs were generated from RB1+/+ and RB1+/- OAMSCs derived from retinoblastoma patients. RB1+/+ and RB1+/- iPSCs were subjected to a step-wise retinal differentiation protocol. Retinal differentiation was evaluated by Real-time PCR and flow cytometry analysis of the retinal markers. To gain further insights into the molecular differences in RB1+/- retinal organoids, a high throughput RNA sequencing followed by differential gene expression analysis and gene set enrichment analysis (GSEA) was performed. The analysis revealed a shift from the regular metabolic process of glycolysis to oxidative phosphorylation in the RB1+/- retinal organoids. To investigate further, we performed assays to determine the levels of pyruvate, lactate and ATP in the retinal organoids. The results revealed significant increase in ATP and pyruvate levels in RB1+/- retinal organoids of day 120 compared to that of the RB1+/+. The results thus revealed enhanced ATP production in the RB1+/- retinal organoids. CONCLUSION: The study provides novel insights into the metabolic phenotype of heterozygous RB1 mutant suggesting dysregulation of energy metabolism and glycolytic pathways to be first step even before the changes in cellular proliferation or other phenotypic consequences ensue.
Assuntos
Trifosfato de Adenosina , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Organoides , Retina , Proteínas de Ligação a Retinoblastoma , Retinoblastoma , Ubiquitina-Proteína Ligases , Humanos , Trifosfato de Adenosina/metabolismo , Diferenciação Celular/genética , Glicólise/genética , Heterozigoto , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Mutação/genética , Organoides/metabolismo , Retina/metabolismo , Retina/citologia , Retinoblastoma/genética , Retinoblastoma/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Protein tyrosine phosphatase receptor type E (PTPRE) is a member of the "classical" protein tyrosine phosphatase subfamily and regulates a variety of cellular processes in a tissue-specific manner by antagonizing the function of protein tyrosine kinases. PTPRE plays a tumorigenic role in different human cancer cells, but its role in retinoblastoma (RB), the most common malignant eye cancer in children, remains to be elucidated. Etoposide-resistant RB cell lines and RB patients display significant higher PTPRE expression levels compared to chemosensitive counterparts and the healthy human retina, respectively. PTPRE promotor methylation analyses revealed that PTPRE expression in RB is not regulated via this mechanism. Lentiviral PTPRE knockdown (KD) induced a significant decrease in growth kinetics, cell viability, and anchorage-independent growth of etoposide-resistant Y79 and WERI RB cells. Caspase-dependent apoptosis rates were significantly increased and a re-sensitization for etoposide could be observed after PTPRE depletion. In vivo chicken chorioallantoic membrane (CAM) assays revealed decreased tumor formation capacity as well as reduced tumor size and weight following PTPRE KD. Expression levels of miR631 were significantly downregulated in etoposide-resistant RB cells and patients. Transient miR631 overexpression resulted in significantly decreased PTPRE levels and concomitantly decreased proliferation and increased apoptosis levels in etoposide-resistant RB cells. These impacts mirror PTPRE KD effects, indicating a regulation of PTPRE via this miR. Additionally, PTPRE KD led to altered phosphorylation of protein kinase SGK3 and-dependent on the cell line-AKT and ERK1/2, suggesting potential PTPRE downstream signaling pathways. In summary, these results indicate an oncogenic role of PTPRE in chemoresistant retinoblastoma.
Assuntos
Apoptose , Resistencia a Medicamentos Antineoplásicos , Etoposídeo , Neoplasias da Retina , Retinoblastoma , Humanos , Retinoblastoma/metabolismo , Retinoblastoma/genética , Retinoblastoma/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Animais , Apoptose/efeitos dos fármacos , Etoposídeo/farmacologia , Etoposídeo/uso terapêutico , Neoplasias da Retina/metabolismo , Neoplasias da Retina/genética , Neoplasias da Retina/patologia , Neoplasias da Retina/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Transdução de Sinais/efeitos dos fármacos , MasculinoRESUMO
Retinoblastoma (RB) is a type of pediatric solid tumor in the fundus. The lack of precision therapies combined with the difficulty of delivering small interfering RNA (siRNA) into the eyes means that there is currently no nucleic acid-based therapy for RB in clinical practice. Here, we reported on anti-GD2 and glutathione-responsive spherical nucleic acids (SNAs), loaded with siRNA and the inhibitor NVP-CGM097, which jointly blocked the oncogenic factor n in RB cells (Y79 and WERI-RB-1). The SNAs were formed through the self-assembly of bifunctional cholesterol amphiphiles containing aptamers that specifically targeted GD2-positive RB cells, allowing for the formation of an SNA with a dense DNA shell. The aptamer/siRNA component functioned both as a carrier and a payload, enhancing the specific recognition and delivery of both components and constituting an active agent for MDM2 regulation. Following SNA endocytosis by RB cells, siRNA and NVP-CGM097 were released from the SNA particles by glutathione, which synergistically blocked the MDM2-p53 pathway, increasing p53 protein content and inducing cell apoptosis. This study showed a potent antitumor effect following intravitreal injection of SNAs in Y79 tumor-bearing mice through clinical manifestation and tumor pathological analysis. In hematological analysis and hepatotoxicity assays, SNAs were safer for mice than melphalan, the favored drug for treating RB in clinical practice. Our results illustrated the potential of intravitreally injected SNAs as a precision medicine for treating RB.
Assuntos
Aptâmeros de Nucleotídeos , Proteínas Proto-Oncogênicas c-mdm2 , RNA Interferente Pequeno , Retinoblastoma , Animais , Humanos , Camundongos , Apoptose/efeitos dos fármacos , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Neoplasias da Retina/tratamento farmacológico , Neoplasias da Retina/patologia , Neoplasias da Retina/metabolismo , Neoplasias da Retina/genética , Retinoblastoma/tratamento farmacológico , Retinoblastoma/patologia , Retinoblastoma/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos ICR , FemininoRESUMO
Complete loss of RB1 causes retinoblastoma. Here, we report the generation of three RB1-/- iPSC lines using CRISPR/Cas9 based editing at exon 18 of RB1 in a healthy control hiPSC line. The edited cells were clonally expanded, genotyped and characterized to establish the mutant lines. Two of the mutant lines are compound heterozygous, with different in-del mutations in each of their alleles, while the third mutant line is homozygous, with identical edits in both alleles. All lines maintained their stemness, pluripotency, formed embryoid bodies with cell types of all three lineages, displayed a normal karyotype and lost RB1 expression.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neoplasias da Retina , Retinoblastoma , Humanos , Retinoblastoma/genética , Retinoblastoma/metabolismo , Sistemas CRISPR-Cas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ligação a Retinoblastoma/genéticaRESUMO
In childhood, retinoblastoma (RB) is the most common primary tumor in the eye. Long term therapeutic management with etoposide of this life-threatening condition may have diminishing effectiveness since RB cells can develop cytostatic resistance to this drug. To determine whether changes in receptor-mediated control of Ca2+ signaling are associated with resistance development, fluorescence calcium imaging, semi-quantitative RT-qPCR analyses, and trypan blue dye exclusion staining patterns are compared in WERI-ETOR (etoposide-insensitive) and WERI-Rb1 (etoposide-sensitive) cells. The cannabinoid receptor agonist 1 (CNR1) WIN55,212-2 (40 µM), or the transient receptor potential melastatin 8 (TRPM8) agonist icilin (40 µM) elicit similar large Ca2+ transients in both cell line types. On the other hand, NGF (100 ng/mL) induces larger rises in WERI-ETOR cells than in WERI-Rb1 cells, and its lethality is larger in WERI-Rb1 cells than in WERI-ETOR cells. NGF and WIN55,212-2 induced additive Ca2+ transients in both cell types. However, following pretreatment with both NGF and WIN55,212-2, TRPM8 gene expression declines and icilin-induced Ca2+ transients are completely blocked only in WERI-ETOR cells. Furthermore, CNR1 gene expression levels are larger in WERI-ETOR cells than those in WERI-Rb1 cells. Therefore, the development of etoposide insensitivity may be associated with rises in CNR1 gene expression, which in turn suppress TRPM8 gene expression through crosstalk.
Assuntos
Receptor de Fator de Crescimento Neural , Neoplasias da Retina , Retinoblastoma , Canais de Cátion TRPM , Humanos , Linhagem Celular , Etoposídeo/farmacologia , Etoposídeo/uso terapêutico , Proteínas de Membrana/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Retinoblastoma/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Receptor CB1 de Canabinoide/metabolismoRESUMO
Retinoblastoma is a pediatric intraocular cancer caused by biallelic inactivation of RB1 gene in retinal progenitor cells. Here, we report the generation of a patient-specific induced pluripotent stem cell (iPSC) line (LVPEIi002-A) from a patient diagnosed with retinoblastoma and showing familial inheritance of a nonsense mutation (c.1735C > T) within exon 18 of one of the two alleles. This RB1+/- iPSC line, LVPEIi002-A was generated by reprogramming the peri-orbital fat tissue derived mesenchymal cells and was stably expanded and characterized. It maintains the stemness, pluripotency, normal karyotype, and forms embryoid bodies comprising of all three lineage committed progenitor cells.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neoplasias da Retina , Retinoblastoma , Criança , Humanos , Retinoblastoma/genética , Retinoblastoma/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Retina/metabolismo , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ligação a Retinoblastoma/genéticaRESUMO
Retinoblastoma (RB) is the most prevalent ocular tumor of childhood, and its extraocular invasion significantly increases the risk of metastasis. Nevertheless, a single-cell characterization of RB local extension has been lacking. Here, we perform single-cell RNA sequencing on four RB samples (two from intraocular and two from extraocular RB patients), and integrate public datasets of five normal retina samples, four intraocular samples, and three extraocular RB samples to characterize RB local extension at the single-cell level. A total of 128,454 qualified cells are obtained in nine major cell types. Copy number variation inference reveals chromosome 6p amplification in cells derived from extraocular RB samples. In cellular heterogeneity analysis, we identified 10, 8, and 7 cell subpopulations in cone precursor like cells, retinoma like cells, and MKI67+ photoreceptorness decreased (MKI67+ PhrD) cells, respectively. A high expression level of SOX4 was detected in cells from extraocular samples, especially in MKI67+ PhrD cells, which was verified in additional clinical RB samples. These results suggest that SOX4 might drive RB local extension. Our study presents a single-cell transcriptomic landscape of intraocular and extraocular RB samples, improving our understanding of RB local extension at the single-cell resolution and providing potential therapeutic targets for RB patients.
Assuntos
Neoplasias da Retina , Retinoblastoma , Humanos , Retinoblastoma/metabolismo , Variações do Número de Cópias de DNA , Neoplasias da Retina/genética , Neoplasias da Retina/patologia , Perfilação da Expressão Gênica , Fatores de Transcrição SOXC/genéticaRESUMO
RB1 deficiency leads to retinoblastoma (Rb), the most prevalent intraocular malignancy. Tumor-associated macrophages (TAMs) are related to local inflammation disorder, particularly by increasing cytokines and immune escape. Microglia, the unique resident macrophages for retinal homeostasis, are the most important immune cells of Rb. However, whether RB1 deficiency affects microglial function remain unknown. In this study, microglia were successfully differentiated from Rb patient- derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), and then we investigated the function of RB1 in microglia by live imaging phagocytosis assay, immunofluorescence, RNA-seq, qRT-PCR, ELISA and retina organoids/microglia co-culturing. RB1 was abundantly expressed in microglia and predominantly located in the nucleus. We then examined the phagocytosis ability and secretion function of iMGs in vitro. We found that RB1 deficiency did not affect the expression of microglia-specific markers or the phagocytic abilities of these cells by live-imaging. Upon LPS stimulation, RB1-deficient microglia displayed enhanced innate immune responses, as evidenced by activated MAPK signaling pathway and elevated expression of IL-6 and TNF-α at both mRNA and protein levels, compared to wildtype microglia. Furthermore, retinal structure disruption was observed when retinal organoids were co-cultured with RB1-deficient microglia, highlighting the potential contribution of microglia to Rb development and potential therapeutic strategies for retinoblastoma.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neoplasias da Retina , Retinoblastoma , Humanos , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Microglia/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Retina , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologiaRESUMO
OBJECTIVE: Retinoblastoma (RB) is a prevalent type of eye cancer in youngsters. Prospero homeobox 1 (Prox1) is a homeobox transcriptional repressor and downstream target of the proneural gene that is relevant in lymphatic, hepatocyte, pancreatic, heart, lens, retinal, and cancer cells. The goal of this study was to investigate the role of Prox1 in RB cell proliferation and drug resistance, as well as to explore the underlying Notch1 mechanism. METHODS: Human RB cell lines (SO-RB50 and Y79) and a primary human retinal microvascular endothelial cell line (ACBRI-181) were used in this study. The expression of Prox1 and Notch1 mRNA and protein in RB cells was detected using quantitative real time-polymerase chain reaction (RT-qPCR) and Western blotting. Cell proliferation was assessed after Prox1 overexpression using the Cell Counting Kit-8 and the MTS assay. Drug-resistant cell lines (SO-RB50/vincristine) were generated and treated with Prox1 to investigate the role of Prox1 in drug resistance. We employed pcDNA-Notch1 to overexpress Notch1 to confirm the role of Notch1 in the protective function of Prox1. Finally, a xenograft model was constructed to assess the effect of Prox1 on RB in vivo. RESULTS: Prox1 was significantly downregulated in RB cells. Overexpression of Prox1 effectively decreased RB cell growth while increasing the sensitivity of drug-resistant cells to vincristine. Notch1 was involved in Prox1's regulatory effects. Notch1 was identified as a target gene of Prox1, which was found to be upregulated in RB cells and repressed by increased Prox1 expression. When pcDNA-Notch1 was transfected, the effect of Prox1 overexpression on RB was removed. Furthermore, by downregulating Notch1, Prox1 overexpression slowed tumor development and increased vincristine sensitivity in vivo. CONCLUSION: These data show that Prox1 decreased RB cell proliferation and drug resistance by targeting Notch1, implying that Prox1 could be a potential therapeutic target for RB.