S-Adenosylmethionine Inhibits the Proliferation of Retinoblastoma Cell Y79, Induces Apoptosis and Cell Cycle Arrest of Y79 Cells by Inhibiting the Wnt2/ß-Catenin Pathway.

Retinoblastoma is one of the most common primary intraocular malignancies in young children. Traditional treatment methods such as chemotherapy often come with significant adverse effects, such as hearing loss, cognitive impairment, and vision loss. Therefore, there is an urgent need to explore a novel therapeutic drug that is both effective and safe. S-adenosylmethionine (SAM) is a natural compound known to exhibit anti-proliferative effects in various cancer cell lines. However, to date, no studies investigated the effects of SAM on retinoblastoma cells and its potential mechanisms of action. Therefore, this study aims to investigate the impact of SAM on retinoblastoma cells and explore its possible mechanisms of action, with the hope of providing new insights into the treatment of this disease. The optimal concentration of SAM was determined using the Cell Counting Kit-8 assay. The effect of SAM on retinoblastoma proliferation was assessed using the 5-ethynyl-2'-deoxyuridine cell proliferation assay. Y79 cells were subjected to hematoxylin and eosin stain and electron microscopy to observe any morphological changes induced by SAM. The stages of SAM's action on the retinoblastoma cell cycle and its apoptotic effects were measured using flow cytometry. The apoptotic effect of SAM on retinoblastoma was further confirmed using the TUNEL assay. Differential expression of related genes was detected through RT-PCR. In vivo subcutaneous tumor formation in nude mice and immunohistochemistry were employed to validate the effect of SAM on retinoblastoma-related phenotypes. Western blotting was conducted to investigate whether SAM modulated retinoblastoma-related phenotypes via the Wnt2/ß-catenin pathway. SAM arrested the cell cycle of retinoblastoma at the G1 phase, induced apoptosis of retinoblastoma cells through the Wnt2/ß-catenin pathway, and affected their morphology and even ultrastructure. In addition, in vitro and in vivo experiments demonstrated that SAM had an oncogenic effect on retinoblastoma. In this study, we verify in vitro and in vivo whether SAM inhibits the proliferation of retinoblastoma cell Y7, induces apoptosis and cell cycle arrest of Y79 cells by inhibiting the Wnt2/ß-catenin pathway, and affects the morphology and structure of retinoblastoma cell Y79.

Craniospinal irradiation and/or intraventricular radioimmunotherapy after high-dose chemotherapy and autologous stem cell rescue in patients with CNS retinoblastoma-Safety and outcomes.

BACKGROUND: The prognosis for patients with central nervous system (CNS) retinoblastoma (RB) (trilateral or stage 4b metastatic RB) treated with high-dose chemotherapy and autologous stem cell transplant (HDC-ASCT) remains poor. The impact of irradiation when administered as part of upfront therapy post HDC-ASCT on treatment outcomes and survival is unknown. METHODS: We performed a retrospective review of all patients with CNS RB (seven stage 4b, eight trilateral, one pineal lesion belonging to methylation group RB) who underwent induction chemotherapy with an intent to proceed to HDC-ASCT at two institutions. RESULTS: Twelve of 16 patients (n = 75%) achieved an objective response to induction chemotherapy, while four patients had progressive/refractory disease; two patients responded to subsequent therapy and proceeded to ASCT, and two patients did not. Seven of 14 patients who underwent HDC-ASCT, received radiotherapy as part of upfront therapy post HDC-ASCT in the form of craniospinal irradiation (CSI) (n = 3), intraventricular radioimmunotherapy (n = 3), or both CSI and intraventricular radioimmunotherapy (n = 1). The Kaplan-Meier estimate of overall survival for these patients was 62.5% at 5 years; no patients developed second malignant neoplasms within the radiation fields. For the seven patients who did not receive radiotherapy, the overall survival was 28.6% at 5 years. CONCLUSIONS: CSI (23.4 Gy) alone or in conjunction with intraventricular RIT may have clinical utility in eliminating persistent MRD post HDC-ASCT, contributing to improved disease-free survival in patients with CNS RB. This treatment strategy merits evaluation in a prospective, multicenter clinical trial for patients with CNS metastatic RB.

Retinoblastoma in Ceara: An epidemiological study in a Brazilian pediatric oncology referral center.

PURPOSE: Although Brazil has a high prevalence of retinoblastoma, there is a lack of epidemiological data on the disease. Thus, in this study, we aimed to evaluate the epidemiological profile of patients diagnosed with retinoblastoma in the ophthalmology department of a pediatric tertiary referral hospital in Ceara, Brazil. METHODS: A descriptive and cross-sectional study was conducted by retrospectively analyzing the clinical and socioeconomic data from the medical records of pediatric patients followed-up at the hospital between 2007 and 2021. Retinoblastoma was diagnosed on the basis of a fundoscopic or histopathologic examination. RESULTS: The data of 105 patients were included in the study, and the mean patient age at the time of diagnosis was 1.7 years. Most of the patients were women (50.5%) and hailed from rural areas (57.4%), which was associated with a higher tumor stage. Of the 150 patients, 57.1% initially presented with leukocoria. Ocular hyperemia was associated with more advanced stages of retinoblastoma (p=0.004). Bilateral involvement was observed in 25.7% of the patients and at a significantly younger age (p=0.009). The presence of retinal detachment, vascularized lesions, and vitreous seeds significantly increased the likelihood of requiring enucleation. DISCUSSION: This study presents an epidemiological description of retinoblastoma in Brazil, which highlights the significance of early detection. Delayed diagnosis is associated with a poorer visual prognosis and higher mortality rate, particularly in patients with unilateral disease. Risk factors for a more severe disease were retinal detachment, vascularized lesions, and vitreous seeds. The correlation between histopathological features and clinical outcomes was limited. CONCLUSION: Further studies are required to assess the influence of ocular hyperemia, fundoscopic assessment, and histopathologic findings on the prognosis of retinoblastoma. Moreover, it is critical to devise interventions to reduce the time-to-diagnosis in rural areas.

Outcome of Retinoblastoma After Implementation of National Retinoblastoma Treatment Guidelines in South Africa.

PURPOSE: Retinoblastoma, a curable childhood cancer, has been identified as a tracer cancer in the WHO Global Initiative for Childhood Cancer. To document the outcomes of children with retinoblastoma in South Africa, treated as per the first prospective standard national treatment guidelines for childhood cancer in South Africa. PATIENTS AND METHODS: All children diagnosed with retinoblastoma between 2012 and 2016 in five South African pediatric oncology units were treated with a standard treatment on the basis of the International Society of Pediatric Oncology-Pediatric Oncology in Developing Countries guidelines for high-income settings. Treatment included focal therapy with/without chemotherapy, or enucleation with/without chemotherapy, and orbital radiotherapy, depending on enucleated eye histology. The end point was survival at 24 months, using Kaplan-Meier curves with log-rank (Mantel-Cox) and chi-square (χ2) tests with respective P values reported. RESULTS: A total of 178 children were included in the study; 68% presented with unilateral disease. The median age was 27 months (range 0-118 months) with a male:female ratio of 1:0.75. The overall survival was 79% at 24 months with significant association with stage at diagnosis (P < .001) and older age over 2 years as opposed to younger than 2 years (P < .001). Causes of death were disease progression/relapses in 90% (34 of 38) and unknown in 2% (1 of 38), whereas treatment abandonment was 1.7% (3 of 178). CONCLUSION: Efficacy with national treatment guidelines was confirmed, and feasibility of implementing standard national childhood cancer treatment guidelines was documented, involving multidisciplinary teams in South Africa. Outcome was significantly associated with stage at diagnosis and age.

Injectable Dendrimer Hydrogel Delivers Melphalan in Both Conjugated and Free Forms for Retinoblastoma.

We report the successful synthesis of an injectable dendrimer hydrogel (DH) carrying melphalan, a clinical drug for retinoblastoma treatment, in both conjugated and free forms. Polyamidoamine (PAMAM) dendrimer generation 5 (G5) is surface-modified with an acid-sensitive acetal-dibenzocyclooctyne linker and then undergoes azide-alkyne cycloaddition with melphalan-PEG-N3 conjugate to form G5-acetal-melphalan. During the DH gelation between G5-acetal-melphalan and PEG-diacrylate, free melphalan is added, resulting in a hydrogel (G5-acetal-melphalan-DH/melphalan) that carries the drug in both conjugated and free forms. Melphalan is slowly released from G5-acetal-melphalan-DH/melphalan, with the conjugated melphalan released more quickly at pH 5.3 due to acid-triggered acetal bond cleavage. The formulation's in vitro safety and efficacy were established on human corneal epithelia (HCE-2) and retinoblastoma cells (Y79). In an in vivo Y79 tumor xenograft model of retinoblastoma, intratumorally injected G5-melphalan-DH formulation prolonged tumor suppression. This injectable, multimodal, pH-responsive formulation shows promise for intravitreal injection to treat retinoblastoma.

PRMT1 mediates the proliferation of Y79 retinoblastoma cells by regulating the p53/p21/CDC2/cyclin B pathway.

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.

EIF4A3-induced hsa_circ_0078136 inhibits the tumorigenesis of retinoblastoma via IL-17 signaling pathway.

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.

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.

A retrospective study on adjuvant chemotherapy in retinoblastoma: validation of the new recommendation against treatment for pT2a tumors based on the 8th AJCC classification.

BACKGROUND: Retinoblastoma (RB) is an intraocular malignant tumor detected in early childhood with variable global impact. Histopathological classification of the tumor in enucleated globes with RB is the key for the decision of adjuvant chemotherapy use. We aim to validate the use of adjuvant chemotherapy in cases with combined pre-laminar/intralaminar optic nerve (ON) invasion and focal choroidal invasion according to the American Joint Committee on Cancer (AJCC) 8th classification. METHODS: This is a retrospective study conducted at King Abdulaziz University Hospital (KAUH) and King Khalid Eye Specialist Hospital (KKESH) in Riyadh, Saudi Arabia of all RB cases who underwent enucleation over 22 years (2000 to 2021). The histopathological findings were reviewed to identify the enucleated globes classified as pT2a tumors, as an inclusion criterion. Basic demographic and clinical data were collected via chart review Simple descriptive and basic statistical analysis of the data was used where applicable. RESULTS: Thirty-one patients who had an enucleated globe with RB that fit into the above classification were included. Sixteen were males and 15 were females. The median age was 14 months (IQR = 14 months). Most of the patients (93.5%) had no family history of RB. The commonest presentation was leukocoria in 87.1% followed by squint in 32.3%. Fourteen patients (45.2%) were treated by enucleation alone while 17 patients (54.8%) received adjuvant chemotherapy. Out of these, 7 patients had unilateral RB and the remaining 10 patients had bilateral RB. None of our patients developed recurrence or metastatic disease irrespective of the indication for adjuvant chemotherapy use after a maximum period of follow up reaching 17.84 years and a median of 10.6 years (IQR = 5.92). CONCLUSIONS: In patients with 8th AJCC histopathological classification of pT2a, chemotherapy following enucleation might not be justified. The outcome in our untreated group of patients did not differ from the treated group with the absence of metastasis after a relatively long period of follow up with a median exceeding 10 years in both groups. Therefore, the risk and benefit of post enucleation adjuvant chemotherapy in the treatment of unilateral RB should be carefully decided and discussed with the primary caregivers taking into consideration the most recent evidence and recommendations in the literature.

Investigating druggable kinases for targeted therapy in retinoblastoma.

Retinoblastoma (RB) is a childhood retinal neoplasm and commonly treated with cytotoxic chemotherapeutic agents. However, these therapeutic approaches often lead to diverse adverse effects. A precise molecular therapy will alleviate these side effects and offer better treatment outcomes. Over the years, kinases have become potential drug targets in cancer therapy. Hence, we aimed to investigate genetic alterations of putative kinase drug targets in RB. Targeted exome sequencing was performed on 35 RB tumors with paired blood samples using a gene panel consisting of 29 FDA-approved kinase genes. Single nucleotide variants were analyzed for pathogenicity using an in-house pipeline and copy number variations (CNVs) were detected by a depth of coverage and CNVPanelizer. The correlation between genetic changes and clinicopathological features was assessed using GraphPad Prism. Three somatic mutations, two in ERBB4 and one in EGFR were identified. Two of these mutations (ERBB4 c.C3836A & EGFR c.A1196T) were not reported earlier. CNV analysis revealed recurrent gains of ALK, MAP2K2, SRC, STK11, and FGFR3 as well as frequent losses of ATM, PI3KCA and ERBB4. Notably, nonresponsive tumors had a higher incidence of amplifications in clinically actionable genes such as ALK. Moreover, ALK gain and ATM loss were strongly correlated with optic nerve head invasion. In conclusion, our study revealed genetic alterations of druggable kinases in RB, providing preliminary insights for the exploration of kinase-targeted therapy in RB.

Study on the role of MYCN in retinoblastoma by inhibiting p53 and activating wnt/ßcatenin/Fra-1 signaling pathway by reducing DKK3.

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.

Comprehensive identification of pathogenic variants in retinoblastoma by long- and short-read sequencing.

Retinoblastoma (RB) is the most common intraocular malignancy in childhood. The causal variants in RB are mostly characterized by previously used short-read sequencing (SRS) analysis, which has technical limitations in identifying structural variants (SVs) and phasing information. Long-read sequencing (LRS) technology has advantages over SRS in detecting SVs, phased genetic variants, and methylation. In this study, we comprehensively characterized the genetic landscape of RB using combinatorial LRS and SRS of 16 RB tumors and 16 matched blood samples. We detected a total of 232 somatic SVs, with an average of 14.5 SVs per sample across the whole genome in our cohort. We identified 20 distinct pathogenic variants disrupting RB1 gene, including three novel small variants and five somatic SVs. We found more somatic SVs were detected from LRS than SRS (140 vs. 122) in RB samples with WGS data, particularly the insertions (18 vs. 1). Furthermore, our analysis shows that, with the exception of one sample who lacked the methylation data, all samples presented biallelic inactivation of RB1 in various forms, including two cases with the biallelic hypermethylated promoter and four cases with compound heterozygous mutations which were missing in SRS analysis. By inferring relative timing of somatic events, we reveal the genetic progression that RB1 disruption early and followed by copy number changes, including amplifications of Chr2p and deletions of Chr16q, during RB tumorigenesis. Altogether, we characterize the comprehensive genetic landscape of RB, providing novel insights into the genetic alterations and mechanisms contributing to RB initiation and development. Our work also establishes a framework to analyze genomic landscape of cancers based on LRS data.

Retinoblastoma caused by an RB1 variant with unusually low penetrance in a Danish family.

Retinoblastoma is the most common eye cancer in children. It is caused by pathogenic alterations of both alleles of the tumor suppressor gene RB1. In heritable retinoblastoma, a constitutional RB1 variant predisposes the cells to tumor formation, and loss of the other allele is a prerequisite for the development of retinoblastoma. Heritable retinoblastoma is inherited in an autosomal dominant manner; however, the majority of cases are the result of a de novo pathogenic RB1 variant. Penetrance is usually high (>90%), but with marked inter-familial variability. In some families, penetrance is incomplete and family members who develop tumors tend to remain unilaterally affected. Moreover, some families with low penetrance also show a parent-of-origin effect. We describe a patient with unilateral retinoblastoma caused by a previously unreported likely pathogenic RB1 variant (c.1199T>C) that disrupts a highly conserved amino acid residue within the A-box functional domain. Segregation analysis showed that the variant had unusually low penetrance as nine non-affected family members carried the same variant. We emphasize the use of genetic analysis on tumor DNA for classifying the RB1 variant, and underline the challenges in clinical management and counseling of families carrying the specific RB1 variant.

Evolving criteria for group E retinoblastoma: impact on outcomes.

OBJECTIVE: To assess the impact of evolving criteria for group E retinoblastoma on ocular survival outcomes. DESIGN: A retrospective observational study. METHODS: Single-institution consecutive case series of patients with advanced intraocular retinoblastoma (groups D and E) were classified based on International Intraocular Retinoblastoma Classification (IIRC) and International Classification of Retinoblastoma (ICRB) criteria. The main outcomes measured were ocular survival, frequency of histopathologic risk factors (HRF), and the need for adjuvant therapy. RESULTS: A total of 332 eyes of 298 patients were classified into group D (150, 45%) and E eyes (182, 55%) based on IIRC criteria. ICRB classification resulted in upstaging of 57 group D eyes (17%) to group E. Eyes that were upstaged to group E from D in the ICRB classification (E1) differed significantly, with a greater proportion undergoing primary enucleation (17 of 57, 30%) than those that were not (10 of 93, 11%) (p = 0.003). Similar significant differences were observed between group E2 and E3 eyes (p < 0.0001). Ocular survival according to Kaplan-Meier estimates at 12 months of 79%, 59%, 49%, and 1% differed significantly between all groups (ICRB D, E1, E2, and E3, respectively). CONCLUSION: Proposed new subgrouping of group E eyes into E1, E2, and E3 based on clinical criteria is based upon natural history of tumor progression and is predictive of ocular survival. Preservation of the existing lower boundaries for group E by ICRB and IIRC offers the possibility of reanalyzing existing published data.

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.