Exploring the utility of human DNA methylation arrays for profiling mouse genomic DNA

AbstractIllumina Infinium Human Methylation (HM) BeadChips are widely used for measuring genome-scale DNA methylation, particularly in relation to epigenome-wide association studies (EWAS) studies. The methylation profile of human samples can be assessed accurately and reproducibly using the HM27 BeadChip (27,578 CpG sites) or its successor, the HM450 BeadChip (482,421 CpG sites). To date no mouse equivalent has been developed, greatly hindering the application of this methodology to the wide range of valuable murine models of disease and development currently in existence. We found 1308 and 13,715 probes from HM27 and HM450 BeadChip respectively, uniquely matched the bisulfite converted reference mouse genome (mm9). We demonstrate reproducible measurements of DNA methylation at these probes in a range of mouse tissue samples and in a murine cell line model of acute myeloid leukaemia. In the absence of a mouse counterpart, the Infinium Human Methylation BeadChip arrays have utility for methylation profiling in non-human species

The RNA Binding Protein Quaking Regulates Formation of circRNAs

SummaryCircular RNAs (circRNAs), formed by non-sequential back-splicing of pre-mRNA transcripts, are a widespread form of non-coding RNA in animal cells. However, it is unclear whether the majority of circRNAs represent splicing by-products without function or are produced in a regulated manner to carry out specific cellular functions. We show that hundreds of circRNAs are regulated during human epithelial-mesenchymal transition (EMT) and find that the production of over one-third of abundant circRNAs is dynamically regulated by the alternative splicing factor, Quaking (QKI), which itself is regulated during EMT. Furthermore, by modulating QKI levels, we show the effect on circRNA abundance is dependent on intronic QKI binding motifs. Critically, the addition of QKI motifs is sufficient to induce de novo circRNA formation from transcripts that are normally linearly spliced. These findings demonstrate circRNAs are both purposefully synthesized and regulated by cell-type specific mechanisms, suggesting they play specific biological roles in EMT

The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is characterized by the presence of a fusion protein EWS/WT1, arising from the t (11;22) (p13;q12) translocation. Here we examine the oncogenic properties of two splice variants of EWS/WT1, EWS/WT1-KTS and EWS/WT1 + KTS. METHODS: We over-expressed both EWS/WT1 variants in murine embryonic fibroblasts (MEFs) of wild-type, p53+/- and p53-/- backgrounds and measured effects on cell-proliferation, anchorage-independent growth, clonogenicity after serum withdrawal, and sensitivity to cytotoxic drugs and gamma irradiation in comparison to control cells. We examined gene expression profiles in cells expressing EWS/WT1. Finally we validated our key findings in a small series of DSRCT. RESULTS: Neither isoform of EWS/WT1 was sufficient to transform wild-type MEFs however the oncogenic potential of both was unmasked by p53 loss. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 enhanced cell-proliferation, clonogenic survival and anchorage-independent growth. EWS/WT1 expression in wild-type MEFs conferred resistance to cell-cycle arrest after irradiation and daunorubicin induced apoptosis. We show DSRCT commonly have nuclear localization of p53, and copy-number amplification of MDM2/MDMX. Expression of either isoform of EWS/WT1 induced characteristic mRNA expression profiles. Gene-set enrichment analysis demonstrated enrichment of WNT pathway signatures in MEFs expressing EWS/WT1 + KTS. Wnt-activation was validated in cell lines with over-expression of EWS/WT1 and in DSRCT. CONCLUSION: In conclusion, we show both isoforms of EWS/WT1 have oncogenic potential in MEFs with loss of p53. In addition we provide the first link between EWS/WT1 and Wnt-pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterize DSRCT

Direct transcriptional regulation by nuclear microRNAs

Abstract not availableMarika Salmanidis, Katherine Pillman, Gregory Goodall, Cameron Bracke

The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is characterized by the presence of a fusion protein EWS/WT1, arising from the t (11;22) (p13;q12) translocation. Here we examine the oncogenic properties of two splice variants of EWS/WT1, EWS/WT1-KTS and EWS/WT1 + KTS. METHODS: We over-expressed both EWS/WT1 variants in murine embryonic fibroblasts (MEFs) of wild-type, p53+/- and p53-/- backgrounds and measured effects on cell-proliferation, anchorage-independent growth, clonogenicity after serum withdrawal, and sensitivity to cytotoxic drugs and gamma irradiation in comparison to control cells. We examined gene expression profiles in cells expressing EWS/WT1. Finally we validated our key findings in a small series of DSRCT. RESULTS: Neither isoform of EWS/WT1 was sufficient to transform wild-type MEFs however the oncogenic potential of both was unmasked by p53 loss. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 enhanced cell-proliferation, clonogenic survival and anchorage-independent growth. EWS/WT1 expression in wild-type MEFs conferred resistance to cell-cycle arrest after irradiation and daunorubicin induced apoptosis. We show DSRCT commonly have nuclear localization of p53, and copy-number amplification of MDM2/MDMX. Expression of either isoform of EWS/WT1 induced characteristic mRNA expression profiles. Gene-set enrichment analysis demonstrated enrichment of WNT pathway signatures in MEFs expressing EWS/WT1 + KTS. Wnt-activation was validated in cell lines with over-expression of EWS/WT1 and in DSRCT. CONCLUSION: In conclusion, we show both isoforms of EWS/WT1 have oncogenic potential in MEFs with loss of p53. In addition we provide the first link between EWS/WT1 and Wnt-pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterize DSRCT

Akt1 is the principal Akt isoform regulating apoptosis in limiting cytokine concentrations

The activation of the Akt signalling in response to cytokine receptor signalling promotes protein synthesis, cellular growth and proliferation. To determine the role of Akt in interleukin-3 (IL-3) signalling, we generated IL-3-dependent myeloid cell lines from mice lacking Akt1, Akt2 or Akt3. Akt1 deletion resulted in accelerated apoptosis at low concentrations of IL-3. Expression of constitutively active Akt1 was sufficient to delay apoptosis in response to IL-3 withdrawal, but not sufficient to induce proliferation in the absence of IL-3. Akt1 prolonged survival of Bim- or Bad-deficient cells, but not cells lacking Puma, indicating that Akt1-dependent repression of apoptosis was in part dependent on Puma and independent of Bim or Bad. Our data show that a key role of Akt1 during IL-3 signalling is to repress p53-dependent apoptosis pathways, including transcriptional upregulation of Puma. Moreover, our data indicate that regulation of BH3-only proteins by Akt is dispensable for Akt-dependent cell survival

Development of a Modifiable Nutrition Algorithm Addressing Nutrition Guidelines in Paediatric Oncology: a Report From the International Pediatric Oncology Nutrition Group

This journal suppl. entitled: Supplement: SIOP Abstratcs: 45th Congress of the International Society of Paediatric Oncology (SIOP) ... 2013Poster Session - Liver tumours: abstract no. P-0235PURPOSE/OBJECTIVE: The five hospitals of Hong Kong forming the Hong Kong Paediatric Haematology and Oncology Study Group (HKPHOSG) established a uniform treatment protocol for hepatoblastoma (HB) since 1996 comprising of primary surgical resection for resectable tumour or in most instances, neoadjuvant chemotherapy (cisplatinum, 5-fluorouracil and vincristine or alternative regimen of cisplatinum and doxorubicin) followed by complete surgical resection of tumour. We reviewed the population-based treatment results of HB in Hong Kong. MATERIALS AND METHODS: The clinical data of all cases diagnosed between 1-Jun-1996 and 31-Dec-2011 were reviewed up to the date of 31-Dec-2012. Each case was assigned as standard risk (SR) or high risk (HR) according to the following criteria, HR: presence of either AFP < ? 100 ng/ml or unresectable tumour or tumour rupture or distant metastases or portal vein thrombosis; SR: resectable tumour without the HR features. RESULTS: During the study period, a total of 51 cases of HB including 35 SR and 16 HR cases were diagnosed. Twenty-eight (55%) were males. The median ages of the whole group, SR and HR were 1.2 years (range, 0.1 to 14.6 years), 1.0 year (0.1 to 14.6 years) and 1.2 years (0.3 to 11.3 years), respectively. The median follow up duration was 3.7 years (range, 0.0 to 15.8 years). The 5-year event-free survival (EFS) and overall survival (S) of the whole group were 74.5% and 79.9%, respectively. The 5-year EFS and S of the SR cases were 93.4% and 100% whilst those of the HR cases were 36.5% and 41.7%, respectively (p < 0.0001 for SR v HR). CONCLUSIONS: The treatment results of the SR HB are significantly better than those of the HR cases. Future goals may aim at reducing the chemotherapy for the SR cases and improving the survival of the HR cases.Link_to_subscribed_fulltex

Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion

Published online 28.07.2014The microRNAs of the miR‐200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago‐HITS‐CLIP technology for transcriptome‐wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR‐200a and miR‐200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR‐200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR‐200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho‐ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR‐200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration.Cameron P Bracken, Xiaochun Li, Josephine A Wright, David M Lawrence, Katherine A Pillman, Marika Salmanidis, Matthew A Anderson, B Kate Dredge, Philip A Gregory, Anna Tsykin, Corine Neilsen, Daniel W Thomson, Andrew G Bert, Joanne M Leerberg, Alpha S Yap, Kirk B Jensen, Yeesim Khew‐Goodall, Gregory J Goodal