Retinoblastoma Cell Lines Research Articles

Hypericin and Naringenin Exert no Significant Synergistic Apoptotic Effect on Y79 Retinoblastoma Cell Line.

According to the anti-cancer impact of hypericin and naringenin, we put the main aim of this study to unravel the apoptotic/anti-cancer effect of these compounds on Y79 retinoblastoma cell line. To calculate the 50%inhibitory concentration (IC50) of hypericin for 24 and 48 hours, XTT assay performed. Cytotoxic effect of naringenin investigated by XTT and trypan blue exclusion assay further confirmed the inhibitory impact of these agents on Y79 cells viability. Flow cytometry Annexin V/PI determined the cell death. The mRNAs expression level of Bax and Bcl-2 investigated by real-time PCR in different groups including the control, cells treated with naringenin, hypericin, or concurrent with both compounds. The 24 and 48 hours IC50 of hypericin, calculated to be 2.5 and 1.25 (μg/ml), respectively. 50 (μg/ml) naringenin induced about 20% and 30% apoptosis in Y79 cells after 24 and 48 hours. Trypan blue staining and flow cytometry confirmed this data. Moreover, flowcytometry results, revealed that the kind cell death occurred in these cells post treatment was mostly apoptosis. Simultaneous treatment with both agents didn't show synergistic effect. Bax/Bcl-2 ratio increased in cells treated with hypericin but in cells treated with narigenin didn't show significant increase in the Bax mRNA level. Hypericin had more cytotoxic effect in Y79 cells compared with naringenin. Furthermore, hypericin and naringenin didn't have apoptotic synergistic effect in these cells. According to the real-time PCR results, hypericin induces apoptosis in Y79 cells by disrupt the ratio of Bax/Bcl-2.

METTL14-Mediated m6a Modification of CDKN2A Promotes the Development of Retinoblastoma by Inhibiting the p53 Pathway.

The methyltransferase 14, N6-adenosine-methyltransferase subunit (METTL14) and Cyclin-dependent kinase inhibitor 2A (CDKN2A) have been identified as involved in the regulation of various cancer progression, while their mechanism and regulatory effect in retinoblastoma (RB) is still unclear. Cell colony formation, CCK-8 as well as Western blotting were used to evaluate the proliferation, apoptosis as well as p53 protein level of RB cell line. The METTL14 and CDKN2A levels were detected by qRT-PCR or Western blotting when METTL14 was up-regulated or CDKN2A was down-regulated. MeRIP and Pearson analysis were performed to confirm the regulatory relationship between METTL14 among CDKN2A. We found that the levels of CDKN2A and METTL14 were abundant in RB samples, as well as RB cells. METTL14 enhances N6-methyladenosine (m6A) modification of CDKN2A to upregulate its mRNA and protein levels. The proliferation of RB cells can be inhibited by silencing CDKN2A, which promotes apoptosis and p53 protein level. Furthermore, high-expression of METTL14 eliminated the anti-tumor effect of CDKN2A silencing in RB progression in vitro. CDKN2A is mediated by METTL14-m6A modified and restrains p53 pathway activation to accelerate the malignancy of RB. This points to the METTL14-m6A-CDKN2A-p53 pathway axis as a possible prospective target for the future RB treatment.

CTSB Nuclear Translocation Facilitates DNA Damage and Lysosomal Stress to Promote Retinoblastoma Cell Death

Retinoblastoma (RB) is a pernicious tumor originating from photoreceptor precursor cells that often endangers the lives of children. The purpose of our study was to further investigate the influence of cathepsin B (CTSB) nuclear translocation on RB cell death. Y79 cells were injected into the vitreous cavity of nude mice at a dose of 4 µL/mouse to establish an animal model of RB. Real-time quantitative polymerase chain reaction (RT–qPCR), Western blot analysis, a comet assay, a Cell Counting Kit-8 (CCK-8) assay and flow cytometry were used to measure the levels of the interrelated genes and proteins and to evaluate alterations in autophagy, apoptosis, proliferation, DNA damage and cell cycle arrest. CTSB was found to be expressed at low levels in RB animal model samples and RB cell lines. Functionally, CTSB nuclear translocation promoted DNA damage, cell cycle arrest, ferroptosis and autophagy in Y79 cells and inhibited their proliferation. Downstream mechanistic studies showed that nuclear translocation of CTSB facilitates DNA damage and cell cycle arrest in RB cells by inhibiting breast cancer 1 protein (BRCA1) expression and also activates the signal transducer and activator of transcription 3/stimulator of interferon response cGAMP interactor 1 (STAT3/STING1) pathway to induce lysosomal stress, leading to ferroptosis and autophagy in Y79 cells and alleviating RB. Nuclear translocation of CTSB facilitates DNA damage and cell cycle arrest in RB cells by inhibiting BRCA1 expression and activating the STAT3/STING1 pathway and induces lysosomal stress, which eventually leads to ferroptosis and autophagy and mitigates RB.

Retinoblastoma vulnerability to combined de novo and salvage pyrimidine ribonucleotide synthesis pharmacologic blockage

Retinoblastoma is an eye cancer that commonly affects young children. Despite significant advances, current treatments cause side effects even when administered locally, and patients may still have to undergo enucleation. This is particularly disheartening in cases of bilateral retinoblastoma. Hence, there is an urgent need for novel therapeutic strategies. Inhibitors of the enzyme dihydroorotate dehydrogenase (DHODH), which is involved in the de novo pyrimidine ribonucleotide synthesis pathway, have proven to be effective in preclinical trials against several cancers including pediatric cancers. Here we tested whether blocking pyrimidine ribonucleotide synthesis promotes retinoblastoma cell death. Cultured retinoblastoma cell lines were treated with small molecule inhibitors of DHODH alone or in combination with inhibitors of nucleoside uptake to also block the salvage pathway for pyrimidine ribonucleotide formation. On their own, DHODH inhibitors had a moderate killing effect. However, the combination with nucleoside uptake inhibitors greatly enhanced the effect of DHODH inhibition. In addition, we observed that pyrimidine ribonucleotide synthesis blockage can cause cell death in a p53 mutant retinoblastoma cell line derived from a patient with metastasis. Explaining these results, the analysis of a published patient cohort revealed that loss of chr16q22.2 (containing the DHODH gene) is amongst the most frequent alterations in retinoblastoma and that these tumors often show gains in chromosome regions expressing pyrimidine ribonucleotide salvage factors. Furthermore, these genome alterations associate with malignancy. These results indicate that targeting pyrimidine ribonucleotide synthesis may be an effective therapeutic strategy to consider as a treatment for retinoblastoma.

A simplified protocol to induce hypoxia in a standard incubator: A focus on retinal cells

Hypoxia chambers have traditionally been used to induce hypoxia in cell cultures. Cellular responses to hypoxia can also be mimicked with the use of chemicals such as cobalt chloride (CoCl2), which stabilizes hypoxia-inducible factor alpha-subunit proteins. In studies of ocular cells using primary cells and cell lines, such as Müller glial cell (MGC) lines, photoreceptor cell lines, retinal pigment epithelial (RPE) cell lines and retinoblastoma cell lines oxygen levels employed in hypoxia chambers range typically between 0.2% and 5% oxygen. For chemical induction of hypoxic response in these cells, the CoCl2 concentrations used typically range from 100 to 600 μM. Here, we describe simplified protocols for stabilizing cellular hypoxia-inducible factor-1α (HIF-1α) in cell culture using either a hypoxia chamber or CoCl2. In addition, we also provide a detailed methodology to confirm hypoxia induction by the assessment of protein levels of HIF-1α, which accumulates in response to hypoxic conditions. Furthermore, we provide a summary of conditions applied in previous studies of ocular cells.

NSUN2-mediated m5 C RNA methylation dictates retinoblastoma progression through promoting PFAS mRNA stability and expression.

The precise temporal and spatial regulation of N5 -methylcytosine (m5 C) RNA modification plays essential roles in RNA metabolism, and is necessary for the maintenance of epigenome homeostasis. Howbeit, the mechanism underlying the m5 C modification in carcinogenesis remains to be fully addressed. Global and mRNA m5 C levels were determined by mRNA isolation and anti-m5 C dot blot in both retinoblastoma (RB) cells and clinical samples. Orthotopic intraocular xenografts were established to examine the oncogenic behaviours of RB. Genome-wide multiomics analyses were performed to identify the functional target of NSUN2, including proteomic analysis, transcriptome screening and m5 C-methylated RNA immunoprecipitation sequencing (m5 C-meRIP-seq). Organoid-based single-cell analysis and gene-correlation analysis were performed to verify the NSUN2/ALYREF/m5 C-PFAS oncogenic cascade. Herein, we report that NSUN2-mediated m5 C RNA methylation fuels purine biosynthesis during the oncogenic progression of RB. First, we discovered that global and mRNA m5 C levels were significantly enriched in RBs compared to normal retinas. In addition, tumour-specific NSUN2 expression was noted in RB samples and cell lines. Therapeutically, targeted correction of NSUN2 exhibited efficient therapeutic efficacy in RB both in vitro and in vivo. Through multiomics analyses, we subsequently identified phosphoribosylformylglycinamidine synthase (PFAS), a vital enzyme in purine biosynthesis, as a downstream candidate target of NSUN2. The reintroduction of PFAS largely reversed the inhibitory phenotypes in NSUN2-deficient RB cells, indicating that PFAS was a functional downstream target of NSUN2. Mechanistically, we found that the m5 C reader protein ALYREF was responsible for the recognition of the m5 C modification of PFAS, increasing its expression by enhancing its RNA stability. Conclusively, we initially demonstrated that NSUN2 is necessary for oncogenic gene activation in RB, expanding the current understanding of dynamic m5 C function during tumour progression. As the NSUN2/ALYREF/m5 C-PFAS oncogenic cascade is an important RB trigger, our study suggests that a targeted m5 C reprogramming therapeutic strategy may be a novel and efficient anti-tumour therapy approach.

Corrigendum to “Comprehensive characterization of RB1 mutant and MYCN amplified retinoblastoma cell lines” [Exp. Cell Res. 375 (2019) 92–99

Corrigendum to “Comprehensive characterization of RB1 mutant and MYCN amplified retinoblastoma cell lines” [Exp. Cell Res. 375 (2019) 92–99

Abstract 2557: Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma

Abstract Retinoblastoma is the most common intraocular cancer of childhood, with approximately 8,000 new cases each year worldwide. While survival rates are relatively high in developed countries, death can occur in up to half of children in some regions. In addition, blindness is common in patients who survive, and improved therapies are clearly needed. While loss of RB gene function is essential for the initiation of almost all retinoblastoma, additional molecular changes driving their growth and dissemination are poorly understood. Improved understanding of the molecular pathways driving retinoblastoma growth and dissemination may allow us to develop more effective and less toxic therapies. Alterations leading to BCL-6 corepressor (BCOR) loss of function are the second most common mutations in retinoblastoma after changes affecting the RB locus, and are found in more aggressive tumors. BCOR mutations are also associated with metastasis and poor prognosis in other types of pediatric cancer, suggesting that targeting it will represent a new therapeutic approach for aggressive retinoblastoma. Here, we found that BCOR knockdown or knockout in two retinoblastoma cell lines with high baseline levels of protein caused significantly increased invasion/migration, proliferation and clonogenicity. In contrast, overexpression of BCOR in retinoblastoma cells with low/no baseline expression showed opposite effects. In orthotopic xenograft models, BCOR knockout dramatically increased the size and spread of retinoblastoma xenografts as compared to parental controls. In order to determine the molecular pathways modulated by BCOR, we used RNA sequencing and found that loss of function induces IGF1 transcription, resulting in activation of IGF1R and the ERK signaling pathway. Pharmacological inhibition of IGF1/IGF1R signaling using Lincitinib or AEW541 inhibited proliferation and migration in BCOR knockout retinoblastoma cells in vitro, and Lincitinib dramatically slowed xenograft growth in vivo, suggesting that induction of IGF1 represents an important downstream effector following BCOR loss. Our studies support a functional role for BCOR mutations in aggressive retinoblastoma, and suggest that this is mediated at least in part by increased IGF1R signaling, which represents a new therapeutic target for these malignant childhood tumors of the eye. Citation Format: Su-Chan Lee, Lujain Alaali, Charles G. Eberhart. Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2557.

Abstract 2004: Selective cytotoxicity of multiple myeloma using novel cell membrane lipid-extracted nanoliposomes

Abstract Background: Multiple Myeloma (MM) is a highly malignant hematologic disease that originates in the bone marrow from tumorigenic plasma cells that rapidly proliferate and often metastasize before detection or symptom manifestation. Due to the enhanced anaerobic metabolism of cancer vis-a-vis normal cells, elevated media levels of LDH following cell death can serve as a reliable benchmark for in-vitro targeted MM treatment efficacy. Cell membrane Lipid Extracted Nanoliposomes (CLENs) are novel drug delivery vehicles that demonstrate preferential uptake by neoplastic cells. By varying the composition of CLENs, they can be tailored to optimize liposomal uptake by MM cells. This study evaluates the degree to which optimized in-vitro cellular uptake of CLENs translates to cytotoxicity by comparing the attrition rate of doxorubicin hydrochloride (DOX)-loaded MM CLENs compared to relevant liposomal and cellular controls. Methods: The RPMI 8226 cell line was this study’s cellular model for MM. RPMI-8226 cells were seeded at 10,000 cells/mL in 48 well plates. The Y79 (retinoblastoma) cell line served as the negative control for all in vitro experiments. Both cell lines were exposed to two different liposome preparations loaded with 3 mol% doxorubicin hydrochloride (DOX), a MM targeted CLENs preparation of 95% DOPC and 5 mol% RPMI-8226 cell lipid extract (LE) and a non-specific, 100 % DOPC liposome. Liposome preparations were separated from free drug by centrifugation and overnight dialysis. Following overnight liposome exposure, the CyQUANTTM LDH Cytotoxicity Assay Kit (Invitrogen) was utilized to assess extracellular LDH activity and evaluate cytotoxicity of DOX-loaded RPMI-8226 LE in vitro. Results: The mean diameter for the DOX-loaded DOPC (100%) and DOPC/LE (95/5) nanoliposomes was 203 ± 24.3 nm (n=4) and 235 ± 24.6 nm (n=4), respectively. The average zeta potential was -15.79 ± 0.33 mV (n=2) and 0.26 ± 0.19 mV (n=2) for DOPC (100%) and DOPC/LE (95/5), respectively. The mean percent of doxorubicin incorporated into DOPC (100%) and DOPC/LE (95/5) was 72% (n=4) and 84% (n=4), respectively. Following overnight exposure of RPMI 8226 cells to DOX-loaded DOPC (100%) and DOX-loaded DOPC/LE (95/5) at 50 nM and 150 nM, preliminary results showed that the inclusion of LE significantly improved cytotoxicity when compared to the DOX-loaded DOPC control. Additionally, DOX-loaded DOPC/LE (95/5) displayed enhanced extracellular LDH activity compared to the DOX-loaded DOPC control, indicated by higher red formazan fluorescence intensity. There was no difference in LDH activity or cytotoxicity between the two DOX-loaded preparations against Y79 (negative control) cells. Conclusions: Preliminary results suggest that RPMI 8226-CLENs represent an improvement over the use of more conventional nanoliposome preparations. Future studies will evaluate additional drug-loaded CLENs in vitro. Citation Format: Matthew Amorin, Peter Daley, William Smullen, Robert Campbell. Selective cytotoxicity of multiple myeloma using novel cell membrane lipid-extracted nanoliposomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2004.

Abstract 3541: Tumoroid-based screening platform to test focal, chemo- and combination therapy for retinoblastoma

Abstract Current treatments for retinoblastoma, the most common intraocular malignancy, rely on a limited number of drugs repurposed from other pediatric cancer therapies. Drug toxicity and relapse of the disease necessitate new therapeutic strategies. The implementation of drug candidates in the clinic is usually followed by in vitro validation, which requires accurate and clinically relevant in vitro models. We report here a robust 3D tumoroid-based platform, for testing various clinical protocols, e.g. therapeutic agents following a single or repeated exposure, alone or in combination with focal therapy (thermotherapy). The platform, generated from the Y79 retinoblastoma cell line, consists of matrix-embedded tumoroids, which retain the key features of the disease and allow prolonged culture time. Following the optimization of growth conditions, the effect of single and repeated exposure to chemotherapeutic agents has been measured by high-throughput automated fluorescence imaging followed by quantification. The clinical conditions of chemothermotherapy have been recapitulated with a setup consisting of: an infrared diode laser (810 nm, 0.3 W) that heats the tumoroids in the presence of indocyanine green (50 µg/mL); a thermal camera to enable real-time temperature measurement; and an environment-controlled inverted microscope to ensure physiological conditions and precise focusing of the laser. Melphalan and carboplatin, two drugs currently used to treat retinoblastoma in clinical protocols of chemotherapy and chemothermotherapy respectively, have been applied to validate the system.Tumoroid viability and size progressively decreased over 4 weeks of repeated drug exposure with melphalan, reflecting the response of retinoblastoma in advanced clinical cases. Carboplatin treatment showed an activity enhancement in the tumoroid model similar to that observed in the clinic, when combined with focal therapy, demonstrating that the platform system closely recapitulates the clinical conditions. Inversely, the thermotherapy experimental protocol designed for 2D in vitro cell culture and commonly used to date, i.e. an incubation temperature increased to 42 °C for 1 h, did not show an improvement of the carboplatin cytotoxicity, confirming that our laser-based thermotherapy protocol provides a more accurate and clinically relevant drug response. Such an approach, combining physiologically relevant in vitro cancer models and investigation of drugs in well-established treatment modalities, can significantly reduce the number of new drug candidates that give false-positives or overestimated efficacies when evaluated in relevant in vivo models. It is expected that the platform will contribute to the implementation of new therapeutic strategies to treat retinoblastoma, and could also be adapted to other diseases for which repeated drug exposure and/or chemothermotherapy is relevant. Citation Format: Irina Sinenko, Fabien Kuttler, Valentin Simeonov, Alexandre Moulin, Christina Stathopoulos, Gerardo Turcatti, Adeline Berger, Francis Munier, Paul Dyson. Tumoroid-based screening platform to test focal, chemo- and combination therapy for retinoblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3541.

Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma.

The chemotherapy of retinoblastoma (RB), a malignant ocular childhood disease, is often limited by the development of resistance against commonly used drugs. We identified inositol polyphosphate 4-phosphatase type II (INPP4B) as a differentially regulated gene in etoposide-resistant RB cell lines, potentially involved in the development of RB resistances. INPP4B is controversially discussed as a tumor suppressor and an oncogenic driver in various cancers, but its role in retinoblastoma in general and chemoresistant RB in particular is yet unknown. In the study presented, we investigated the expression of INPP4B in RB cell lines and patients and analyzed the effect of INPP4B overexpression on etoposide resistant RB cell growth in vitro and in vivo. INPP4B mRNA levels were significantly downregulated in RB cells lines compared to the healthy human retina, with even lower expression levels in etoposide-resistant compared to the sensitive cell lines. Besides, a significant increase in INPP4B expression was observed in chemotherapy-treated RB tumor patient samples compared to untreated tumors. INPP4B overexpression in etoposide-resistant RB cells resulted in a significant reduction in cell viability with reduced growth, proliferation, anchorage-independent growth, and in ovo tumor formation. Caspase-3/7-mediated apoptosis was concomitantly increased, suggesting a tumor suppressive role of INPP4B in chemoresistant RB cells. No changes in AKT signaling were discernible, but p-SGK3 levels increased following INPP4B overexpression, indicating a potential regulation of SGK3 signaling in etoposide-resistant RB cells. RNAseq analysis of INPP4B overexpressing, etoposide-resistant RB cell lines revealed differentially regulated genes involved in cancer progression, mirroring observed in vitro and in vivo effects of INPP4B overexpression and strengthening INPP4B's importance for cell growth control and tumorigenicity.

Effects of long non-coding RNA MALAT1-targeting miR-570-3p and miR-34a on the invasion, proliferation, and apoptosis of human retinoblastoma cells

Purpose: To investigate the expression of lncRNA MALAT1-targeting miR-570-3p and miR-34a and its effects on the invasion, proliferation, and apoptosis of human retinoblastoma cells.
 Methods: MiR-34a, miR-570-3p, and IncRNA MALAT1 in a human normal retinal vascular endothelial cell line (ACBRI-181), human retinoblastoma cell line (SO-Rb50), human normal retinal tissue and human retinoblastoma tissue were determined. Luciferase assay was used to verify the targeting relationship between LncRNA MALAT1 and miR-570-3p and miR-34a. while cell invasion, cell apoptosis and cell proliferation were assessed by Transwell assay, flow cytometry, and tumor pellet-forming assay, respectively.
 Results: LncRNA MALAT1 in SO-Rb50 cell line and human retinoblastoma tissue line were significantly up-regulated, while the expression levels of miR-34a and miR-570-3p were significantly down-regulated (p < 0.05). Luciferase assay results showed that lncRNA MALAT1 targeted miR-570-3p and miR-34a. The invasion and proliferation of SO-Rb50 cells in the miR-570-3p inhibitor and miR-34a inhibitor groups were significantly increased, while the apoptosis of SO-Rb50 cells was significantly decreased (p < 0.05). However, the invasion and proliferation of SO-Rb50 cells in sh-MALAT1 group were significantly decreased, while apoptosis significantly increased. However, compared with sh-MALAT1 group alone, the invasion and proliferation of SO-Rb50 cells in co-transfected sh-MALAT1+miR-570-3p inhibitor + miR-34a inhibitor group w significantly increased, but apoptosis significantly decreased (p < 0.05).
 Conclusion: LncRNA MALAT1 negatively regulates miR-570-3p and miR-34a to promote the invasion and proliferation of human retinoblastoma SO-Rb50 cells and inhibit apoptosis. These findings may be of significance in developing suitable therapies for human retinoblastoma

Bioactive Polymeric Nanoparticles of Moringa oleifera Induced Phyto-Photothermal Sensitization for the Enhanced Therapy of Retinoblastoma

Treatment of retinoblastoma is limited due to its delayed detection and inaccesbility of drugs to reach the retina crossing the blood-retinal barrier. With the advancements in nanotechnology, photothermal therapy (PTT) employing plasmonic nanomaterials and/or NIR dyes have emerged as an affordable alternative owing to the spatial control that is offered by the modality leading to localized and enhanced therapeutic efficacy with minimal invasiveness. However, the modality is limited in its clinical application owing to the increased heat shock resistance of the tumor cells in response to the heat that is generated via PTT. Hence, in this study, we explore the role of novel biomolecular fraction of Moringa oleifera (DFM) encapsulated within a polymeric nanosystem, for its anti-heat shock protein (HSP) activity. The MO extract was co-encapsulated with NIR sensitizing dye, IR820 into a biodegradable polycaprolactone (PCL) nano-delivery system (PMIR NPs). The photothermal transduction efficacy of PMIR NPs was validated in vitro against retinoblastoma cell lines. The inherent fluorescence of DFM was utilized to evaluate the cellular internalization of the PMIR NPs using fluorescence microscopy and flow cytometry. The overall oxidative protein damage and downregulation of HSP70 expression upon treatment with PMIR NPs and NIR laser irradiation was evaluated using densiometric protein analysis and Western blotting. Overall, the PMIR NPs exhibited excellent anti-cancer activity when combined with PTT with downregulated HSP70 expression against retinoblastoma cells.

The role of EZH2 as a potential therapeutic target in retinoblastoma

Enhancer of zeste homolog 2 (EZH2) has been reported selectively expressed in postnatal human retinoblastoma (RB). While, the contribution of EZH2 in progression of RB and its clinical importance has not been clarified. Here, immunohistochemistry (IHC) was performed on tumor specimens from 53 RB patients. UNC1999 and GSK503, inhibitors targeting EZH2, were incubated with human RB cell line WERI-Rb-1 and Y79 to assess the role and mechanism of EZH2 in RB proliferation, metastasis and tumor glycolysis. Administration of UNC1999 in subcutaneous tumor model of RB was conducted. The results showed that highly expressed EZH2 in RB tissues was significantly associated with the poor overall survival. UNC1999 and GSK503 inhibited proliferation, migration, invasion and tumor glycolysis of RB. Results in mouse xenograft model confirmed the inhibitory effect of UNC1999 on tumor growth of RB and the regulation effect of EZH2 to STAT3/FoxO1 signaling pathway. Therefore, EZH2 is rewarding to study as a potential target for anti-RB treatment.

Non-synonymous, synonymous, and non-coding nucleotide variants contribute to recurrently altered biological processes during retinoblastoma progression.

Retinoblastomas form in response to biallelic RB1 mutations or MYCN amplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To determine if nucleotide variants recurrently affect specific biological processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to RB1 mutations, MYCN amplification, and established retinoblastoma somatic copy number alterations, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, non-coding and synonymous variants increased the enrichment significance of each over-represented biological process term. To assess the effects of such mutations, we examined the consequences of a 3' UTR variant of PCGF3 (a BCOR-binding component of Polycomb repressive complex I), dual 3' UTR variants of CDC14B (a regulator of sister chromatid segregation), and a synonymous variant of DYNC1H1 (a regulator of P-body assembly). One PCGF3 and one of two CDC14B 3' UTR variants impaired gene expression whereas a base-edited DYNC1H1 synonymous variant altered protease sensitivity and stability. Retinoblastoma cell lines retained only ~50% of variants detected in tumors and enriched for new variants affecting p53 signaling. These findings reveal potentially important differences in retinoblastoma cell lines and tumors and implicate synonymous and non-coding variants, along with non-synonymous variants, in retinoblastoma oncogenesis.

Magnolol regulates miR-200c-3p to inhibit epithelial–mesenchymal transition and retinoblastoma progression by modulating the ZEB1/E-cadherin axis in vitro and in vivo

BackgroundRetinoblastoma, the most common pediatric intraocular malignancy, can develop during embryogenesis, with most children being diagnosed at 3–4 years of age. Multimodal therapies are typically associated with high levels of cytotoxicity and side effects. Therefore, the development of novel treatments with minimal side effects is crucial. Magnolol has a significant anti-tumor effect on various cancers. However, its antitumor effect on retinoblastoma remains unclear. PurposeThe study aimed to determine the effects of magnolol on the regulation of EMT, migration, invasion, and cancer progression in retinoblastoma and the modulation of miR-200c-3p expression and the Wnt/ zinc finger E-box binding homeobox 1 (ZEB1)/E-cadherin axis in vivo and in vitro. MethodsThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to evaluate magnolol-induced cell toxicity in the Y79 retinoblastoma cell line. Flow cytometry and immunostaining assays were performed to investigate the magnolol-regulated mitochondrial membrane potential and the intracellular and mitochondrial reactive oxygen species levels in Y79 retinoblastoma cells. Orthotopic and subcutaneous xenograft experiments were performed in eight-week-old male null mice to study retinoblastoma progression and metastasis. In situ hybridization and quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays were performed to evaluate the level of the anti-cancer miRNA miR-200c-3p. The mRNA and protein levels of E-cadherin, β-catenin, α-smooth muscle actin (α-SMA), fibronectin-1, and ZEB1 were analyzed using RT-qPCR, immunoblot, immunocytochemistry, and immunohistochemistry assays in vitro and in vivo. ResultsMagnolol increased E-cadherin levels and reduced the activation of the EMT signaling pathway, EMT, tumor growth, metastasis, and cancer progression in the Y79 retinoblastoma cell line as well as in the orthotopic and subcutaneous xenograft animal models. Furthermore, magnolol increased the expression of miR-200c-3p. Our results demonstrate that miRNA-200c-3p inhibits EMT progression through the Wnt16/β-catenin/ZEB1/E-cadherin axis, and the ZEB1 silencing response shows that miR-200c-3p regulates ZEB1-mediated EMT in retinoblastoma. ConclusionMagnolol has an antitumor effect by increasing E-cadherin and miRNA-200c-3p expression to regulate ZEB1-mediated EMT and cancer progression in retinoblastoma. The anti-tumor effect of magnolol by increasing E-cadherin and miRNA-200c-3p expression to regulate ZEB1-mediated EMT and cancer progression in retinoblastoma has been elucidated for the first time.

LncRNA MEG3 attenuates the malignancy of retinoblastoma cells through inactivating PI3K /Akt/mTOR signaling pathway

Retinoblastoma (RB) is the most common neoplasm found in the eye of children. There are increasing interests to develop targeted gene therapy for this disease. This study was performed to investigate the impact of long non-coding RNA (lncRNA) MEG3 on the biological features of RB cells. Vector overexpressing MEG3 was constructed and introduced into two RB cell lines. Transfected RB cells were assessed for proliferation, apoptosis, migration ability, expression levels of important genes in the PI3K/Akt/mTOR signaling pathway using qRT-PCR and Western blot analysis. Xenograft mouse models were constructed to determine the tumorigenicity of RB cells overexpressing MEG3. MEG3 mRNA level was significantly lower in RB cells than in non-cancer cells (p < 0.01). Overexpressing MEG3 resulted in significant reduction in cell proliferation (p < 0.05), migration (p < 0.01) and significant increase in apoptosis (p < 0.01). After overexpressing MEG3, p-PI3K, p-Akt and p-mTOR levels were significantly downregulated (p < 0.01). Furthermore, in the xenograft model, RB cells overexpressing MEG3 generated significantly smaller tumors as compared to RB cells that did not overexpress MEG3 (p < 0.05). Our data suggest that MEG3 increases apoptosis and reduces tumorigenicity of RB cells through inactivating the PI3K/Akt/mTOR pathway. Therefore, MEG3 could be further investigated as a potential new therapeutic agent and target for RB therapy.

MicroRNA-708 suppresses the proliferation, migration, and invasion of human retinoblastoma cells by targeting RAP2B, a member of the RAS oncogene family.

Retinoblastoma generally affects children and causes permanent vision failure or even death. MicroRNAs (miRs) have recently gained much attention during recent years. The miR-708 acts as a tumor suppressor in several human cancers, but the former has not been functionally characterized in human retinoblastoma. The present study was designed to investigate the role of miR-708 in human retinoblastoma. The results showed that miR-708 is significantly (P<0.05) downregulated in retinoblastoma cell lines. MiR-708 overexpression significantly (P<0.05) inhibited retinoblastoma cell growth and proliferation by inducing apoptosis. Furthermore, retinoblastoma cells overexpressing miR-708 exhibited a markedly lower migratory rate and invasiveness compared to negative control cells. The bioinformatics and dual luciferase assay revealed a RAS oncogene family protein, RAP2B, which acts as the regulatory target and functional mediator of the molecular role of miR-708 in retinoblastoma. Together, the present study revealed the tumor suppressor role of miR-708 and pointed to the therapeutic implications of miR-708/RAP2B in the treatment of retinoblastoma.

Zebrafish as an Orthotopic Tumor Model for Retinoblastoma Mimicking Routes of Human Metastasis.

Retinoblastoma (RB) is the most common eye cancer in children that has a high mortality rate when left untreated. Mouse models for retinoblastoma have been established but are time- and cost-intensive. The aim of this work was to evaluate an orthotopic transplantation model of retinoblastoma in zebrafish that also allows for tracking migratory routes and to explore advantages and disadvantages with respect to drug testing. Three fluorescence-labeled retinoblastoma cell lines (RB355, WERI-RB-1, Y79) were injected into the left eye of two-day-old zebrafish, while the un-injected right eye served as control. The migratory trajectories of injected retinoblastoma cells were observed until 8 days post injection (dpi), both in lateral and dorsal view, and measuring fluorescence intensity of injected cells was done for RB355 cells. Time until the onset of migration and routes for all three retinoblastoma cell lines were comparable and resulted in migration into the brain and ventricles of the forebrain, midbrain and hindbrain. Involvement of the optic nerve was observed in 10% of injections with the RB355 cell line, 15% with Y79 cells and 5% with WERI-RB-1 cells. Fluorescence intensity of injected RB355 cells showed an initial increase until five dpi, but then decreased with high variability until the end of observation. The zebrafish eye is well suited for the analysis of migratory routes in retinoblastoma and closely mirrors patterns of retinoblastoma metastases in humans.

MODL-30. AN ENGINEERED TRANSGENIC MOUSE MODEL OF RETINOBLASTOMA

Abstract Retinoblastoma, the most common intraocular tumor of childhood, was recently shown to comprise two major molecular subtypes. Subtype 1 includes most heritable forms, with germline mutation of RB1 and a cone-differentiated state. Subtype 2 is often non-hereditary, exhibits stemness features, increased expression of MYCN and its target genes, and a high risk of metastasis. Relative to subtype 1, subtype 2 tumors have a worse prognosis. To date, no mouse model has been available for studying subtype 2 disease. It is known that SHH pathway proteins including SHH, GLI1 and GLI2 are abnormally elevated in human retinoblastoma. We recently discovered that activation of GLI2 in the developing mouse retina leads to intraocular tumors that exhibit cellular and molecular features resembling human subtype 2 retinoblastoma with aberrant upregulation of MYCN and its target genes. Because MYCN is a downstream target of the SHH pathway, we hypothesize that inhibiting the SHH-GLI2-MYCN signaling axis may effectively treat subtype 2 disease. Using our GLI2 based mouse model of retinoblastoma, we will elucidate mechanisms underlying tumor initiation and progression, and evaluate the therapeutic potential of SHH and GLI2 inhibitors in preventing retinoblastoma. We will validate our mouse model findings using human retinoblastoma cell lines and patient-derived tumor xenografts. Our studies will yield new mechanistic insights into the development of subtype 2 retinoblastoma and identify novel molecular targets for early detection, treatment, and prevention of ocular tumors and associated metastatic disease.