Justina McEvoy

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.

Targeting Oxidative Stress in Embryonal Rhabdomyosarcoma

Rhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.

Subcellular localization and cytoplasmic complex status of endogenous Keap1

Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin‐3‐based ubiquitin ligase, and a regulator of Nrf2 activity through the interaction with Nrf2 Neh2 domain. However, the mechanism(s) of Nrf2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule. Here, we report the generation of a new monoclonal antibody for the detection of endogenous Keap1 molecules. Immunocytochemical analysis of mouse embryonic fibroblasts with the antibody revealed that under normal, unstressed condition, Keap1 is localized primarily in the cytoplasm with minimal amount in the nucleus and endoplasmic reticulum. This subcellular localization profile of Keap1 appears unchanged after treatment of cells with diethyl maleate, an electrophile, and/or Leptomycin B, a nuclear export inhibitor. Subcellular fractionation analysis of mouse liver cells showed similar results. No substantial change in the subcellular distribution profile could be observed in cells isolated from butylated hydroxyanisole‐treated mice. Analyses of sucrose density gradient centrifugation of mouse liver cells indicated that Keap1 appears to form multiprotein complexes in the cytoplasm. These results demonstrate that endogenous Keap1 remains mostly in the cytoplasm, and electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of Keap1.

TNF Counterbalances the Emergence of M2 Tumor Macrophages

Cancer can involve non-resolving, persistent inflammation where varying numbers of tumor-associated macrophages (TAMs) infiltrate and adopt different activation states between anti-tumor M1 and pro-tumor M2 phenotypes. Here, we resolve a cascade causing differential macrophage phenotypes in the tumor microenvironment. Reduction in TNF mRNA production or loss of type I TNF receptor signaling resulted in a striking pattern of enhanced M2 mRNA expression. M2 gene expression was driven in part by IL-13 from eosinophils co-recruited with inflammatory monocytes, a pathway that was suppressed by TNF. Our data define regulatory nodes within the tumor microenvironment that balance M1 and M2 populations. Our results show macrophage polarization in cancer is dynamic and dependent on the balance between TNF and IL-13, thus providing a strategy for manipulating TAMs.

Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma

Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 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.

Constitutive Turnover of Cyclin E by Cul3 Maintains Quiescence

ABSTRACT 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.

Changes in retinoblastoma cell adhesion associated with optic nerve invasion

ABSTRACT 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.

The role of the Rb family during mouse retinal development

In the developing central nervous system, newborn cortical neurons express the proneural bHLH transcription factor Neurogenin2 (Neurog2), then undergo active cell migration before arriving at their final destination and undergoing terminal differentiation. We have identified the zinc finger transcriptional repressor Znf238 to be a downstream target of Neurog2; and preliminary experiments suggest that Znf238 controls the timing of migration and differentiation of newborn cortical neurons. These RNAi experiments suggest that knockdown of Znf238 in cortical progenitors resulted in an arrest of cell migration, as well as inducing their premature neurodifferentiation. Remarkably, luciferase reporter assays for the transcriptional activity of Znf238 revealed its antagonism for signalling through Neurog2-type E-box binding sites. We conclude that Neurog2 coordinates the temporal progression for neurodevelopment through stimulation of a Znf238-dependent negative feedback loop.