A MYCN-driven de-differentiation profile identifies a subgroup of aggressive retinoblastoma.

Retinoblastoma are childhood eye tumors arising from retinal precursor cells. Two distinct retinoblastoma subtypes with different clinical behavior have been described based on gene expression and methylation profiling. Using consensus clustering of DNA methylation analysis from 61 retinoblastomas, we identify a MYCN-driven cluster of subtype 2 retinoblastomas characterized by DNA hypomethylation and high expression of genes involved in protein synthesis. Subtype 2 retinoblastomas outside the MYCN-driven cluster are characterized by high expression of genes from mesodermal development, including NKX2-5. Knockdown of MYCN expression in retinoblastoma cell models causes growth arrest and reactivates a subtype 1-specific photoreceptor signature. These molecular changes suggest that removing the driving force of MYCN oncogenic activity rescues molecular circuitry driving subtype 1 biology. The MYCN-RB gene signature generated from the cell models better identifies MYCN-driven retinoblastoma than MYCN amplification and can identify cases that may benefit from MYCN-targeted therapy. MYCN drives tumor progression in a molecularly defined retinoblastoma subgroup, and inhibiting MYCN activity could restore a more differentiated and less aggressive tumor biology.

Histone Deacetylases in Retinoblastoma.

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.

Aqueous VEGF-A Levels as a Liquid Biopsy Biomarker of Retinoblastoma Vitreous Seed Response to Therapy.

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.

Bmi-1 promotes the proliferation, migration and invasion, and inhibits cell apoptosis of human retinoblastoma cells via RKIP.

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.

Focused cancer pathway analysis revealed unique therapeutic targets in retinoblastoma.

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.

ALYREF/THOC4 expression and cell growth modulation in retinoblastoma.

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 40 % 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.

Role of Protein Tyrosine Phosphatase Receptor Type E (PTPRE) in Chemoresistant Retinoblastoma.

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.

Hyper-N-glycosylated SEL1L3 as auto-antigenic B-cell receptor target of primary vitreoretinal lymphomas.

Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress into primary central nervous system lymphoma (PCNSL). To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed B-cell receptors (BCR) from PVRL patients and tested for binding against human auto-antigens. SEL1L3, a protein with multiple glycosylation sites, was identified as the BCR target in 3/20 PVRL cases. SEL1L3 induces proliferation and BCR pathway activation in aggressive lymphoma cell lines. Moreover, SEL1L3 conjugated to a toxin killed exclusively lymphoma cells with respective BCR-reactivity. Western Blot analysis indicates the occurrence of hyper-N-glycosylation of SEL1L3 at aa 527 in PVRL patients with SEL1L3-reactive BCRs. The BCR of a PVRL patient with serum antibodies against SEL1L3 was cloned from a vitreous body biopsy at diagnosis and of a systemic manifestation at relapse. VH4-04*07 was used in both lymphoma manifestations with highly conserved CDR3 regions. Both BCRs showed binding to SEL1L3, suggesting continued dependence of lymphoma cells on antigen stimulation. These results indicate an important role of antigenic stimulation by post-translationally modified auto-antigens in the genesis of PVRL. They also provide the basis for a new treatment approach targeting unique lymphoma BCRs with ultimate specificity.

Generation and characterization of three CRISPR/Cas9 edited RB1 null hiPSC lines for retinoblastoma disease modelling.

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.

An anti-GD2 aptamer-based bifunctional spherical nucleic acid nanoplatform for synergistic therapy targeting MDM2 for retinoblastoma.

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.

Generation of an induced pluripotent stem cell line (LVPEIi002-A) with heterozygous RB1 mutation using peri-orbital fat derived mesenchymal cells of a patient with inherited retinoblastoma.

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.

The Traces of Dysregulated lncRNAs-Associated ceRNA Axes in Retinoblastoma: A Systematic Scope Review.

PURPOSE: Long non-coding RNAs are an essential component of competing endogenous RNA regulatory axes and play their role by sponging microRNAs and interfering with the regulation of gene expression. Because of the broadness of competing endogenous RNA interaction networks, they may help investigate treatment targets in complicated disorders. METHODS: This study performed a systematic scoping review to assess verified loops of competing endogenous RNAs in retinoblastoma, emphasizing the competing endogenous RNAs axis related to long non-coding RNAs. We used a six-stage approach framework and the PRISMA guidelines. A systematic search of seven databases was done to locate suitable papers published before February 2022. Two reviewers worked independently to screen articles and collect data. RESULTS: Out of 363 records, fifty-one articles met the inclusion criteria, and sixty-three axes were identified in desired articles. The majority of the research reported several long non-coding RNAs that were experimentally verified to act as competing endogenous RNAs in retinoblastoma: XIST/NEAT1/MALAT1/SNHG16/KCNQ1OT1, respectively. At the same time, around half of the studies investigated unique long non-coding RNAs. CONCLUSIONS: Understanding the many features of this regulatory system may aid in elucidating the unknown etiology of Retinoblastoma and providing novel molecular targets for therapeutic and clinical applications.

Enhanced innate responses in microglia derived from retinoblastoma patient-specific iPSCs.

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.

Prox1 Suppresses Proliferation and Drug Resistance of Retinoblastoma Cells via Targeting Notch1.

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.

Toxicity of superparamagnetic iron oxide nanoparticles on retinoblastoma mitochondria.

PURPOSE: Retinoblastoma (RB) is one of the most important cancers in children with a higher rate of prevalence in developing countries. Despite different approaches to the treatment of RB, it seems necessary to discover a new approach to its treatment. Today, mitochondria are recognised as an important target in the treatment of cancer. Superparamagnetic iron oxide nanoparticles (SPIONs) have been studied by researchers due to their important biological effects. METHODS: In this study, the effects of SPIONs on mitochondria isolated from Y79 retinoblastoma cells were investigated. RESULTS: The results showed that SPIONs were able to increase the reactive oxygen species (ROS) level and subsequently damage the mitochondrial membrane and release cytochrome c a as one of the important pro-apoptotic proteins of RB mitochondria. Furthermore, the results indicated a decrease in cell viability and an increase in caspase-3 activity in Y79 retinoblastoma cells. CONCLUSIONS: These events can lead to the killing of cancerous mitochondria. Our results suggest that SPIONs can cause mitochondrial dysfunction and death in RB mitochondria.

Gentiopicroside inhibits retinoblastoma cell proliferation, invasion, and tumorigenesis in nude mice by suppressing the PI3K/AKT pathway.

Retinoblastoma is a prevalent pediatric intraocular tumor. The suppressive effect of gentiopicroside (GPS) has been reported on various tumors. This study sought to determine the effect of GPS on retinoblastoma cell proliferation, apoptosis, invasion, and epithelial-mesenchymal transition (EMT), and tumorigenesis in nude mice. The effect and mechanism of GPS on growth, apoptosis, invasion, and EMT were determined by cell counting kit-8 (CCK-8), western blot, flow cytometry, and transwell assays in retinoblastoma cells. Y79 cells were injected into the vitreous cavity of BALB/c­nude mice to construct a retinoblastoma mouse model. Tumor growth and mouse weight were monitored for sequential 5 weeks. The effect of GPS in vivo was assessed by immunohistochemistry (IHC), terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), and western blot assays. GPS decreased the cell viability of both Y79 and Weri-Rb1 cells with the IC50 of 18.85 µM and 27.57 µM, respectively. Besides, GPS reduced the relative expression of proteins involved in proliferation and EMT, and the number of invading cells, while increased the apoptosis rate and the relative expressions of apoptosis proteins in retinoblastoma cells. Mechanically, GPS decreased the relative protein level of PI3K/AKT pathway, which was then recovered after 740 Y-P was applied. Correspondingly, 740 Y-P reversed the inhibitory effect of GPS on growth, invasion, and EMT, and the increased effect of GPS on apoptosis. Additionally, GPS decreased tumor volume and weight as well as the relative level of Ki-67, VEGF, p-PI3K/PI3K, and p-AKT/AKT, while increased the apoptosis rate in vivo. GPS inhibited retinoblastoma cell proliferation and invasion via deactivating the PI3K/AKT pathway in both cell and animal models.

miR-4529-3p Promotes the Progression of Retinoblastoma by Inhibiting RB1 Expression and Activating the ERK Signaling Pathway.

Retinoblastoma (RB) is a malignant ocular cancer that affects children. Several microRNAs (miRNAs) have been implicated in RB regulation. The present study aimed to investigate the role of miR-4529-3p in RB pathogenesis. Scratch, Transwell, and Cell Counting Kit (CCK)-8 assays were conducted to assess the migratory, invasive, and proliferative abilities of RB cells. The expression levels of miR-4529-3p, RB1, and ERK pathway-related proteins were analyzed using western blotting and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Target relationships were verified using dual-luciferase reporter experiments. A murine RB model was developed to analyze the effects of miR-4529-3p on RB tumor growth in vivo. Our experiments revealed high levels of miR-4529-3p and low levels of RB1 in RB tissues. Functional analyses revealed that the migratory, invasive, and proliferative abilities of RB cells were repressed by miR-4529-3p inhibition. Similarly, p-ERK 1/2 protein levels were suppressed by miR-4529-3p inhibition. Furthermore, downregulation of miR-4529-3p limited tumor growth in vivo. Mechanistically, miR-4259-3p targets RB1. Interestingly, RB1 silencing abrogated the alleviative effects of miR-4529-3p downregulation in RB cells. MiR-4529-3p promotes RB progression by inhibiting RB1 and activating the ERK pathway. This evidence suggests that the miR-4529-3p/RB1 regulatory axis may be a prospective target for RB treatment in clinical settings.

Generation and characterization of two induced pluripotent stem cell lines from conjunctiva of a retinoblastoma patient.

Retinoblastoma (RB) is a common intraocular malignancy mostly caused by variation of the tumour suppressor gene RB1. In this study, we successfully generated two induced pluripotent stem cell (iPSC) lines from an infant with non-heritable RB. Both cell clones exhibited typical iPSC characteristics with normal karyotypes, consistent pluripotency markers expression and the capability of trilineage differentiation.