|
|
|||||||||||||||||||||
|
|
||||||||||||||||||||||
|
Centre for Cancer Research Student ProjectsCancer & Innate Immunity Research ProjectsFunctional analysis of relapse predictive genes in Wilms tumour Wilms tumour (neuroblastoma) is a paediatric cancer of the kidney and is a classic model for the connection between normal development and cancer. Although most (around 80-90%) Wilms tumours are curable with current therapies, a number of the remaining tumours go on to relapse and of these almost half are fatal. Using microarray analysis our lab has discovered a gene signature of four genes which predict which tumours are most likely to relapse. Two of these genes are well characterized in a number of different cancers but one, CEBPb(CAAT-enhancer binding protein beta) has not been characterized in Wilms tumour before. This project aims to study the effects of different isoforms of this protein on tumour development using a specific Wilms tumour cell line (WiT49) developed in our laboratory. The project will use a variety of techniques including: cell culture, siRNA (short interfering RNA) for downregulation of gene expression, cloning into lentiviral vectors and infection into cells for overexpression studies, FACS analysis, mutation analysis of luciferase reporter assays (“promoter bashing”), quantitative (Real Time) RT-PCR, Western blotting, analysis of protein-protein and protein-DNA interactions by chromatin immunoprecipitation (ChIP) and tumour modelling by xenografts of tumour tissue in an animal model. The project is suitable for a PhD student or can be divided into smaller projects suitable for honours students. Clinical sensitivity to Interferon (IFN) is determined by a complex coordination of genetic and environmental factors. We have defined a subset of interferon stimulated genes (ISGs) whose sustained expression correlates with heightened IFN sensitivity in renal carcinoma cells lines and contain transcription factor PLZF binding sites in their promoters. Analysis of gene expression in a cells inducible PLZF expression reveals increased expression of ISGs, depends on PLZF expression. Chromatin Immunoprecipitation assays reveal direct association between PLZF and in silico identified PLZF binding sites in ISG promoters. This project will define mechanisms of IFN signaling to PLZF using biochemical and proteomic approaches, investigate its role in stimulating ISG transcription in vivo using a mouse PLZF knockout model and correlate the expression and functional activity of PLZF in renal cell cancers. Negative regulation of Toll like receptor (TLR) pathways is an important mechanism to safeguard against deleterious effects of over-active or prolonged inflammation. We have found that ligands for a variety of TLRs (TLR-2/6, TLR-3, TLR-4, TLR-7, and TLR-9) trigger rapid accumulation of ATF-3 in mouse macrophages or plasmacytoid dendritic cells (pDCs). In primary macrophages from mice with targeted deletion of the atf3 gene, IL-12 and IL-6 production stimulated by various TLR ligands was significantly enhanced relative to responses in wild-type macrophages. Consistent with a role as a negative regulator, intraperitoneal injection of CpG-ODN in ATF3-KO mice results in enhanced cytokine production and DC maturation in splenocytes following treatment. Thus, ATF3 was defined as a negative regulatory transcription factor for innate responses following TLR activation. This project will ATF3 dysregulation in cancers where altered innate immunity has been implicated in pathogenesis. We have shown that short interfering RNA (siRNA) can be robust activators of innate immunity. This can be attributed to the nature of the 3’ends of the siRNA and their interaction with a signaling RNA helicase RIG-I. This project will further explore the nature of this interaction using biophysical and structural biology techniques and characterize the different pathways activated in both immune and non-immune cells. Rapid cellular responses to environmental signals are often mediated by post-transcriptional modifications of proteins by kinases. The protein kinase R has an established role in regulating proliferation and apoptosis via phosphorylation of the key translation factor, eIF-2alpha. Recent studies in our laboratory reveal PKR also elicits a novel transcriptional response by an, as yet, uncharacterised mechanism. A project exists to decipher the mechanism by which PKR regulates this response. The project involves genetics, biochemistry and cell-based study. The project will investigate aspects of innate immunity, focusing on the serine/threonine protein kinase PKR. The kinase has an established role in regulating translation through interaction with eIF2alpha. We have preliminary data indicating PKR associates with several other proteins and want to explore these putative interactions to validate the association, to determine if the proteins are substrates for the kinase, and to explore consequences of the association in a disease context. The project involves genetics, biochemistry and cell-based study. A project exists to investigate mechanisms in the development of Juvenile diabetes (T1D). Although T1D disease is primarily considered a corollary of aberrant immune function, the mechanisms leading to immunological destruction of β-cells in the pancreas are unknown The project is motivated by the recent realisation that the ifih1 gene may be a causal locus in type 1 diabetes (T1D), and a novel observation made in our laboratory correlating polymorphisms in this gene product with different innate immune responses. This observation identifies a functional consequence for the reported genetic variance within ifih1 that may define an important mechanism in the development of T1D. |