Differential CRX and OTX2 expression in human retina and retinoblastoma

The histogenesis of retinoblastoma tumors remains controversial, with the cell-of-origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone-rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner-Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell-of-origin of retinoblastoma tumors and cells expressing CRX and OTX2.     

Single-cell transcriptome profiling reveals intratumoural heterogeneity and malignant progression in retinoblastoma

Retinoblastoma is a childhood retinal tumour that is the most common primary malignant intraocular tumour. However, it has been challenging to identify the cell types associated with genetic complexity. Here, we performed single-cell RNA sequencing on 14,739 cells from two retinoblastoma samples to delineate the heterogeneity and the underlying mechanism of retinoblastoma progression. Using a multiresolution network-based analysis, we identified two major cell types in human retinoblastoma. Cell trajectory analysis yielded a total of 5 cell states organized into two main branches, and the cell cycle-associated cone precursors were the cells of origin of retinoblastoma that were required for initiating the differentiation and malignancy process of retinoblastoma. Tumour cells differentiation reprogramming trajectory analysis revealed that cell-type components of multiple tumour-related pathways and predominantly expressed UBE2C were associated with an activation state in the malignant progression of the tumour, providing a potential novel “switch gene” marker during early critical stages in human retinoblastoma development. Thus, our findings improve our current understanding of the mechanism of retinoblastoma progression and are potentially valuable in providing novel prognostic markers for retinoblastoma.Subject terms: Eye cancer, Differentiation, Prognostic markers     

Crocin inhibits proliferation and induces apoptosis through suppressing MYCN expression in retinoblastoma

The pathogenetic mechanisms of retinoblastoma are still not yet fully elucidated, putting limits to efficacious treatment. Crocin is the main component of saffron, which exhibits significant antitumorigenic properties. The aim of this paper is to investigate the effect of crocin on retinoblastoma. The effects of crocin on the proliferation of human retinoblastoma cells were determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, cell number assay, and colony formation assay. Cell apoptosis induced by crocin was measured by flow cytometry analysis. Cleaved poly(ADP‐ribose) polymerase and cleaved caspase‐3 were tested by western blot analysis. The expression levels of MYCN were assessed by western blot and quantitative polymerase chain reaction and the stability of MYCN messenger RNA was determined by in vitro RNA degradation assays. We found that crocin significantly inhibited the cell proliferation and clonogenicity and induced cell apoptosis in Y79 and WERI‐RB‐1 cells. In addition, crocin treatment significantly reduced the expression and the stability of MYCN. Besides, overexpression of MYCN rescued the inhibitory effect of crocin in Y79 cells. Our findings suggest that crocin exhibits antitumorigenic effects in human retinoblastoma cell lines through a MYCN‐dependent manner, which may provide guidance to logical therapeutic designs in prevention and treatment of retinoblastoma.     

In vitro differentiation of human retinoblastoma cells into neuronal phenotypes

In order to characterize the cell type(s) of origin of human retinoblastoma cells by immunophenotyping, primary cells from seven retinoblastomas and of the corresponding cell lines (RBL lines), as well as four retinoblastoma (RB) lines established by other groups, were compared with rat and human retina cells, and with the adenovirus E1A-transformed human retinoblast cell line HER-Xho1-CC2. Analyses using monoclonal antibodies (Mabs) RB13-2 and RB21-7, originally raised against prenatal rat brain cells and recognizing neural cell surface antigens expressed in a developmental-stage-dependent manner, and three cell-type-specific Mabs (Q211, M501, Mab directed against vimentin) developed by other groups, gave the following results: (i) Retinoblastomas consist of cells expressing differentiated neuronal phenotypes during cultivation in vitro; (ii) All of the newly established RBL lines express neuronal phenotypes; and (iii) Cell lines such as Y79, which have been propagated in vitro for extended periods, do not express antigens specific for the neuronal pathway and cannot, therefore, be considered phenotypically representative of retinoblastoma cells.     

Retracted: PRC1 gene silencing inhibits proliferation,invasion, and angiogenesis of retinoblastoma cells through the inhibition of the Wnt/β-catenin signaling pathway

Retinoblastoma is an ocular malignancy occurring in childhood. The current study evaluates the ability of silenced PRC1 on retinoblastoma cell proliferation, and angiogenesis via the Wnt/β-catenin signaling pathway. A total of 36 cases of retinoblastoma tissues (n = 36) and normal retinal tissues (n = 10) were selected in the current study. Retinoblastoma cells presenting with the high PRC1 messenger RNA (mRNA) expression were selected among the WERI-Rb-1, HXO-RB44, Y79, SO-Rb50, and SO-Rb70 cells lines, and were transfected with siRNA-PRC1 and LiCl (the activator of the Wnt/β-catenin pathway). The expressions of PRC1, VEGF, Wnt1, β-catenin, CyclinD1, extent of β-catenin, and GSK-3β phosphorylation were evaluated. Cell proliferation, cell-cycle distribution, and cell invasion of retinoblastoma cells were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and Transwell assay. The angiogenesis of retinoblastoma cells was detected by tube formation assay. HXO-RB44 and WERI-Rb-1 cells were selected owing to the highest PRC1 mRNA expression. Meanwhile, PRC2 gene silencing presented lower expression levels of PRC1, VEGF, Wnt1, β-catenin, CyclinD1, extent of β-catenin and GSK-3β phosphorylation, decreased proliferation and invasion abilities, extended G0/G1 phase, and shortened S and G2/M phases of HXO-RB44 and WERI-Rb-1 cells, suggesting the silenced PRC2 inactivated Wnt/β-catenin pathway, so as to further restrain the retinoblastoma cell proliferation, invasion, and angiogenesis. These results support the view that PRC1 gene silencing could suppress the proliferation, and angiogenesis of retinoblastoma cells by repressing the Wnt/β-catenin pathway.     

Long Non-coding RNA FEZF1-AS1 Promotes Growth and Reduces Apoptosis Through Regulation of miR-363-3p/PAX6 Axis in Retinoblastoma

Retinoblastoma is the most common malignancy in children's eyes with high incidence. Long non-coding RNAs (lncRNAs) play important roles in the progression of retinoblastoma. LncRNA FEZF1 antisense RNA 1 (FEZF1-AS1) has been found to stimulate retinoblastoma. However, the mechanism of FEZF1-AS1 underlying progression of retinoblastoma is still unclear. In current study, FEZF1-AS1 was up-regulated in retinoblastoma tissues and cells. FEZF1-AS1 overexpression enhanced retinoblastoma cell viability, promoted cell cycle, and inhibited apoptosis. Conversely, FEZF1-AS1 knockdown reduced cell viability, cycle, and elevated apoptosis. The interaction between FEZF1-AS1 and microRNA-363-3p (miR-363-3p) was confirmed. FEZF1-AS1 down-regulated miR-363-3p and up-regulated PAX6. PAX6 was a target gene of miR-363-3p. EZF1-AS1 promoted retinoblastoma cell viability and suppressed apoptosis via PAX6. Further, we demonstrated that FEZF1-AS1 contribute to tumor formation in vivo. In conclusion, FEZF1-AS1 elevated growth and inhibited apoptosis by regulating miR-363-3p/PAX6 in retinoblastoma, which provide a new target for retinoblastoma treatment.

     

A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N∶2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53^−/− cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.     

Bone morphogenetic protein 4 (BMP4) signaling in retinoblastoma cells

Bone morphogenetic proteins (BMPs) - expressed in the developing retina - are known to be involved in the regulation of cell proliferation and apoptosis in several tumor entities. The objective of this study was to determine the role of the BMP4 pathway in retinoblastoma cells, which are absent in a functional retinoblastoma (RB1) gene. BMP receptors were detected in all retinoblastoma cell lines investigated. A correct transmission of BMP signaling via the Smad1/5/8 pathway could be demonstrated in WERI-Rb1 retinoblastoma cells and application of recombinant human BMP4 resulted in an increase in apoptosis, which to a large extend is caspase independent. Cell proliferation was not affected by BMP4 signaling, although the pRb-related proteins p107 and p130, contributing to the regulation of the same genes, are still expressed. WERI-Rb1 cells exhibit elevated endogenous levels of p21(CIP1) and p53, but we did not detect any increase in p53, p21(CIP1)or p27(KIP1) expression levels. Id proteins became, however, strongly up-regulated upon exogenous BMP4 treatment. Thus, RB1 loss in WERI-Rb1 cells is obviously not compensated for by pRb-independent (e.g. p53-dependent) cell cycle control mechanisms, preventing an anti-proliferative response to BMP4, which normally induces cell cycle arrest.     

Regulation of retinoblastoma protein functions by ectopic expression of human cyclins.

The retinoblastoma susceptibility gene (RB) product, the retinoblastoma protein (pRb), functions as a regulator of cell proliferation. Introduction of the RB gene into SAOS-2 osteosarcoma cells, which lack functional pRb, prevents cell cycle progression. Such growth-suppressive functions can be modulated by phosphorylation of pRb, which occurs via cell cycle-regulated kinases. We show that constitutively expressed cyclins A and E can overcome pRb-mediated suppression of proliferation. pRb becomes hyperphosphorylated in cells overexpressing these cyclins, and this phosphorylation is essential for cyclin A- and cyclin E-mediated rescue of pRb-blocked cells. This suggests that G1 and S phase cyclins can act as regulators of pRb function in the cell cycle by promoting pRb phosphorylation.     

Retinoblastoma teaches a new lesson

In this issue, Ajioka et al. (2007) report a new mouse model of retinoblastoma. They show that retinoblastoma is not driven by uncontrolled expansion of retinal progenitor cells, but rather is the result of cell cycle re-entry and expansion of differentiated horizontal interneurons in the retina.     

High rate of detection of 13q14 deletion mosaicism among retinoblastoma patients (using more extensive methods)

Summary Using a cell population with a high proportion of early mitotic cells and by examining more cells derived from peripheral lymphocytes, we found three cases with a 13q14 deletion mosaicism among fifteen retinoblastoma patients; one with a de novo 13/18 balanced translocation, and another with a monosomy 13(q13»q21.2 or 21.3). The three patients with a 13q14 deletion mosaicism had sporadic retinoblastoma (two had bilateral and one unilateral retinoblastoma). The results indicate that 13q14 deletion mosaicism plays a major role in the etiology of this tumor.     

High trefoil factor 1 (TFF1) expression in human retinoblastoma cells correlates with low growth kinetics, increased cyclin-dependent kinase (CDK) inhibitor levels and a selective down-regulation of CDK6

Trefoil factor family (TFFs) peptides facilitate epithelial restitution, but also effect cell proliferation and apoptosis of normal and various cancer cell lines. In a recent study by our group, TFF2 expression was demonstrated in the murine retina, where it exhibits pro-proliferative and pro-apoptotic effects. In the present study, we investigated the expression and function of TFF peptides in eight human retinoblastoma cell lines. TFF1 was the only TFF peptide expressed at detectable levels in immunoblots of retinoblastoma cells. TFF1 expression levels were highly variable in different retinoblastoma cell lines and negatively correlated with cell growth curves. Recombinant human TFF1 had a negative effect on cell viability and caused a reduction in cell proliferation. Retinoblastoma cell lines with high TFF1 expression levels exhibited a selective down-regulation of cyclin-dependent kinase (CDK) 6, whereas CDK4 and CDK2 seem to be unaffected by TFF1 expression. In immunocytochemical studies, we observed a nuclear co-localization of TFF1 and CDK2 in Cajal bodies (CBs). In high TFF1 expressing human retinoblastoma cell lines CBs were smaller and higher in number compared to retinoblastoma lines with low TFF1 expression, indicating differences in cell cycle status between the different retinoblastoma cell lines. Our data further support the notion for a potential tumor suppressor function of TFF1. The nuclear localization of TFF1 in CBs—considered to play a role in cell cycle progression, potentially acting as a platform for CDK-cyclin function—offers a new impetus in the ongoing search for potential TFF1 interacting proteins.     

Repair of potentially lethal X-ray damage in fibroblasts derived from patients with hereditary and D-deletion retinoblastoma

We examined X-ray induced potentially lethal damage repair (PLDR) in density inhibited plateau phase cultures of six fibroblast strains derived from patients with hereditary retinoblastoma and two patients with D-deletion retinoblastoma and compared them to three normal controls. PLD was measured in hereditary retinoblastoma (7 Gy exposure) and normal cells (7 and 9 Gy exposure) after 24 h repair time. PLD survival curves were performed at 2-9 Gy on six retinoblastoma and three normal control cell strains. Thus, PLDR was compared at equitoxic survival levels as well as after exposure to equal doses of radiation. Some retinoblastoma strains showed normal PLDR whereas others were possibly deficient. Implications of PLDR for susceptibility to radiation-induced and spontaneous tumours in hereditary retinoblastoma patients are discussed.     

Stability of retinoblastoma gene expression determines the tumorigenicity of reconstituted retinoblastoma cells.

Mutational inactivation of the retinoblastoma gene (RB) is an invariant feature of the childhood eye cancer retinoblastoma and of tumor cells derived therefrom. In a previous study, retrovirus-mediated transfer of wild-type RB into cultured retinoblastoma cells resulted in a marked enlargement and reduced growth rate of these cells, as well as loss of their tumorigenic properties in nude mice. It was therefore difficult to separate the proposed growth-suppressing and tumor-suppressing activities of RB protein. Here, we show that clones of RB-reconstituted retinoblastoma cells can be isolated that stably express apparently normal RB protein for at least 20 months of continuous culture. These clones were indistinguishable from nonreconstituted cells by multiple parameters including morphology, growth rate, and cell cycle distribution. Despite similar phenotypes in culture, clones with stable RB expression were uniformly nontumorigenic in nude mice, whereas those that lost such expression regained their tumorigenic properties. These results indicate that the tumorigenicity of these cells is entirely determined by the presence or absence of exogenous RB protein expression and that suppression of tumorigenicity is distinct from inhibition of cellular growth in culture.     

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2^-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. Wesley S. Bond and Lalita Wadhwa are co-first authors.     

LncRNA HOTAIR/miR‐613/c‐met axis modulated epithelial‐mesenchymal transition of retinoblastoma cells

Since lncRNAs could modulate neoplastic development by modulating downstream miRNAs and genes, this study was carried out to figure out the synthetic contribution of HOTAIR, miR‐613 and c‐met to viability, apoptosis and proliferation of retinoblastoma cells. Totally 276 retinoblastoma tissues and tumour‐adjacent tissues were collected, and human retinoblastoma cell lines (ie, Y79, HXO‐Rb44, SO‐Rb50 and WERI‐RB1) were also gathered. Moreover, transfections of pcDNA3.1‐HOTAIR, si‐HOTAIR, miR‐613 mimic, miR‐613 inhibitor, pcDNA3.1/c‐met were performed to evaluate the influence of HOTAIR, miR‐613 and c‐met on viability, apoptosis and epithelial‐mesenchymal transition (EMT) of retinoblastoma cells. Dual‐luciferase reporter gene assay was also arranged to confirm the targeted relationship between HOTAIR and miR‐613, as well as between miR‐613 and c‐met. Consequently, up‐regulated HOTAIR and down‐regulated miR‐613 expressions displayed associations with poor survival status of retinoblastoma patients (P < 0.05). Besides, inhibited HOTAIR and promoted miR‐613 elevated E‐cadherin expression, yet decreased Snail and Vimentin expressions (P < 0.05). Simultaneously, cell proliferation and cell viability were also less‐motivated (P < 0.05). Nonetheless, c‐met prohibited the functioning of miR‐613, resulting in promoted cell proliferation and viability, along with inhibited cell apoptosis (P < 0.05). Finally, HOTAIR was verified to directly target miR‐613, and c‐met was the direct target gene of miR‐613 (P < 0.05). In conclusion, the role of lncRNA HOTAIR/miR‐613/c‐met signalling axis in modulating retinoblastoma cells’ viability, apoptosis and expressions of EMT‐specific proteins might provide evidences for developing appropriate diagnostic and treatment strategies for retinoblastoma.