A novel retinoblastoma therapy from genomic and epigenetic analyses.

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss

Coexpression of Normally Incompatible Developmental Pathways in Retinoblastoma Genesis

It is widely believed that the molecular and cellular features of a tumor reflect its cell of origin and can thus provide clues about treatment targets. The retinoblastoma cell of origin has been debated for over a century. Here, we report that human and mouse retinoblastomas have molecular, cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type-specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Furthermore, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro

Constitutive Turnover of Cyclin E by Cul3 Maintains Quiescence▿ †

Two distinct pathways for the degradation of mammalian cyclin E have previously been described. One pathway is induced by cyclin E phosphorylation and is dependent on the Cul1/Fbw7-based E3 ligase. The other pathway is dependent on the Cul3-based E3 ligase, but the mechanistic details of this pathway have yet to be elucidated. To establish the role of Cul3 in the degradation of cyclin E in vivo, we created a conditional knockout of the Cul3 gene in mice. Interestingly, the biallelic loss of Cul3 in primary fibroblasts derived from these mice results in increased cyclin E expression and reduced cell viability, paralleling the loss of Cul3 protein expression. Cell cycle analysis of viable, Cul3 hypomorphic cells shows that decreasing the levels of Cul3 increases both cyclin E protein levels and the number of cells in S phase. In order to examine the role of Cul3 in an in vivo setting, we determined the effect of deletion of the Cul3 gene in liver. This gene deletion resulted in a dramatic increase in cyclin E levels as well as an increase in cell size and ploidy. The results we report here show that the constitutive degradation pathway for cyclin E that is regulated by the Cul3-based E3 ligase is essential to maintain quiescence in mammalian cells

Analysis of MDM2 and MDM4 Single Nucleotide Polymorphisms, mRNA Splicing and Protein Expression in Retinoblastoma

<div><p>Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic <em>RB1</em> gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX) and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191) was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.</p> </div

Expression of MDM4 in retinoblastoma.

<p><b>A</b>) Structure of the genomic organization of the 11 exons of human <i>MDM4</i>. The exons are to scale as shown but the introns are not to scale. (<b>B</b>) Schematic of the spliced <i>MDM4</i> mRNA that produces the full-length MDM4 protein. The location and identification of Affymetrix gene expression array probesets are shown in red and the location of the real time RT-PCR probe/primer set is shown in blue. (<b>C</b>) Boxplots of the Log₂ expression of each of the 5 probesets that uniquely mapped to <i>MDM4</i> for fetal retina, cell lines, xenografts and 52 primary human retinoblastomas. (<b>D</b>) Real time RT-PCR for MDM2 using Taqman probes as shown in (B) for fetal retina at gestational stage week 20 (FW20), control cell lines (NB1691 and U20S), retinoblastoma cell lines (Y79, Weri1, RB355), primary tumors (SJ33, SJ43, SJ45), and retinoblastoma xenografts (SJ39-X, SJ41-X, SJ42-X and MSK176). Values were normalized to the positive control (U2OS). (<b>E</b>) The same data as shown in (D) but normalized to fetal retina. Each bar is the mean and standard deviation of duplicate experiments. (<b>F</b>) The SAGE data from developing mouse retina shows <i>Mdm4</i> is expressed at significant levels in the developing mouse retina. The gray shaded box represents the limit of detection by SAGE (<2 normalized tags per sample). <i>Gapdh</i> and <i>Gpi1</i> are plotted as ubiquitiously expressed internal controls. (<b>G</b>) Immunoblot of MDM4 (green) protein in a subset of the samples analyzed by real time RT-PCR. Gapdh (red) was used as an internal reference for gel loading and the normalized values are presented below the black and white presentation of the blot for MDM4. The antibody was verified for specificity using a blocking peptide (not shown). Recombinant full length Flag-MDM4 protein was included as a positive control. Multiple bands were detected for MDM4 and were quantitated (below the black and white picture of the blot) and normalized to GAPDH and relative to U2OS cell line.</p

Analysis of miR-191 in retinoblastoma.

<p><b>A</b>) Boxplot of the Log₂ expression for hsa-mir-191 probeset in 6 fetal retina and 16 primary human retinoblastomas showing significantly lower expression. (<b>B</b>) MicroRNA real time-RT-PCR for hsa-mir-191 in fetal retina at gestational week 20, retinoblastoma cell lines (Rb355, Y79, and Weri1), retinoblastoma primary tumors, and orthotopic xenografts. Values are normalized to fetal retina. (<b>C</b>) Scatter plots of the Log₂ expression values for hsa-mir-191 and 4 of the representative 44 mir-191 target genes in fetal retina and retinoblastoma primary tumors. Affymetrix probeset in parenthesis. (<b>D</b>) Scatter plot of the Log₂ expression values for hsa-mir-191 and MDM4 (Affymetrix probeset 236814_at) in retinoblastoma primary tumors. Circled are primary tumors with a C/A genotype.</p

Expression of MDM2 in retinoblastoma.

<p><b>A</b>) Structure of the genomic organization of the 11 exons of human <i>MDM2</i>. The exons are to scale as shown, but the introns are not to scale. <b>B</b>) Schematic of the spliced <i>MDM2</i> mRNA that produces the full-length MDM2 protein. The location and identification of Affymetrix gene expression array probesets are shown in red and the location of the real time RT-PCR probe/primer set is shown in blue. (<b>C</b>) Boxplots of the Log₂ expression of each of the 6 probesets that uniquely mapped to <i>MDM2</i> for fetal retina, cell lines, xenografts and 52 primary human retinoblastomas. In general, genes expressed below Log₂ of 7–8 cannot be detected by SAGE and are below the limit of reliable detection for retinal samples. (<b>D</b>) Real time RT-PCR for <i>MDM2</i> using Taqman probes as shown in (B) for fetal retina at gestational stage week 20 (FW20), a control (NB1691) that expresses high levels of MDM2, and a control (U2OS) that expresses high levels of MDM4, retinoblastoma cell lines (Y79, Weri1, RB355), primary tumors (SJ33, SJ43, SJ45), and retinoblastoma xenografts (SJ39-X, SJ41-X, SJ42-X and MSK176). Values were normalized to the positive control (NB1691). (<b>E</b>) The same data as shown in (D) but normalized to fetal retina. Each bar is the mean and standard deviation of duplicate experiments. (<b>F</b>) The SAGE data from developing mouse retina shows <i>Mdm2</i> is not expressed at significant levels in the developing mouse retina. The gray shaded box represents the limit of detection by SAGE (<2 normalized tags per sample). <i>Gapdh</i> and <i>Gpi1</i> are plotted as ubiquitiously expressed internal controls. (<b>G</b>) Immunoblot of MDM2 (green) protein in a subset of the samples analyzed by real time RT-PCR. Gapdh (red) was used as an internal reference for gel loading and the normalized values are presented below the black and white presentation of the blot for MDM2.</p

Analysis of transcript variants of MDM4 in retinoblastoma.

<p><b>A, B</b>) Genomic organization of MDM4 showing two previously identified splice variants for this gene and the corresponding mRNA. MDM4-S results from skipping of exon 6 and MDM4-A results from skipping of exon 9 (dark gray shading). (<b>C</b>) Domain structure of the full length MDM4 protein with known protein modification sites (phosphorylation (P) and sumoylation (S)) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042739#pone.0042739-Mancini1" target="_blank">[18]</a>). The predicted size is 55 kDa with an observed size on SDS-PAGE of ∼75 kDa. (<b>D</b>) The loss of exon 6 in MDM4-S results in a frameshift after residue 114 and a translational termination after residue 140 (diagonal line). The predicted size of MDM4-S is 15 kDa and the observed size on SDS-PAGE is 27 kDa <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042739#pone.0042739-Mancini1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042739#pone.0042739-Rallapalli3" target="_blank">[27]</a>. (<b>E</b>) The loss of exon 9 in MDM4-A results in an in frame fusion that is missing 50 residues spanning a portion of the acidic domain. (<b>F</b>) Representative reads from transcriptome analysis showing the loss of exon 6 and exon 9 in the retinoblastoma orthotopic xenografts. These alternative spliced transcripts were verified by PCR (<b>H</b>) and Sanger sequencing (<b>I</b>). (<b>G</b>) Representative reads from transcriptome analysis of MDM2 showing very little detection for alternative spliced transcripts.</p

Changes in Retinoblastoma Cell Adhesion Associated with Optic Nerve Invasion▿ †

In the 1970s, several human retinoblastoma cell lines were developed from cultures of primary tumors. As the human retinoblastoma cell lines were established in culture, growth properties and changes in cell adhesion were described. Those changes correlated with the ability of the human retinoblastoma cell lines to invade the optic nerve and metastasize in orthotopic xenograft studies. However, the mechanisms that underlie these changes were not determined. We used the recently developed knockout mouse models of retinoblastoma to begin to characterize the molecular, cellular, and genetic changes associated with retinoblastoma tumor progression and optic nerve invasion. Here we report the isolation and characterization of the first mouse retinoblastoma cell lines with targeted deletions of the Rb family. Our detailed analysis of these cells as they were propagated in culture from the primary tumor shows that changes in cadherin-mediated cell adhesion are associated with retinoblastoma invasion of the optic nerve prior to metastasis. In addition, the same changes in cadherin-mediated cell adhesion correlate with the invasive properties of the human retinoblastoma cell lines isolated decades ago, providing a molecular mechanism for these earlier observations. Most importantly, our studies are in agreement with genetic studies on human retinoblastomas, suggesting that changes in this pathway are involved in tumor progression