DNA methylation analysis of the evolution of Wilms tumour from its precursor nephrogenic rests

Recurrent loss of imprinting at 11p15, paucity of recurrent genetic mutations and associated nephrogenic rests (NR; precursor lesions that resemble embryonic kidney (EK)) implicate aberrant DNA methylation in tumourigenesis of paediatric Wilms tumour (WT) and predict that interrogation of the methylome, rather than the genome, is more likely to reveal tumour-specific biomarkers To test if aberrant DNA methylation is implicated in tumourigenesis, methylome analysis was performed on 36 normal kidney (NK), 22 NR, 36 WT and 4 EK, including 20 matched trios and 34 matched NK-WT pairs, using Illumina 450k arrays. Findings were validated with bisulfite-sequencing and RNA sequencing. This thesis describes the successful identification of changes in methylation that distinguish between tissue types. Through analysis of DNA methylation, NR formation was associated with gain of methylation at developmental loci related to Polycomb target binding sites. Evolution to WT was associated with increase in methylation variability in a subset of WTs (group-1), which also showed common changes in methylation in comparison to their associated NR, including silencing of novel tumour suppressor genes. Group-1 WTs were significantly enriched for bilateral cases whereas those in group-2 showed no differences in methylation compared to their associated NR. Comparison between NK and WT identified three DMRs of genome-wide significance (P<5x10-8) for use as tumour-specific biomarkers. As proof of principle for clinical utility, DMR-2 was successfully used in a case study to monitor tumour burden during treatment in cell-free serum DNA. This thesis concludes that methylation levels vary during WT evolution. As group-1 WT included all bilateral cases, our data suggests that methylation analysis could aid treatment planning in bilateral disease and that some WT may be candidates for epigenetic-modifier therapy. These findings define the first cell-free epigenetic biomarker for WT with potential for clinical utility

Immunotherapy, Tumor Microenvironment and Survival Signaling

The book is based on the Cancers journal Special Issue entitled “Immunotherapy, Tumor Microenvironment and Survival Signaling", and focuses on important problems concerning tumors and tumor microenvironment interactions, as well as novel immunotherapies such as CAR-T cell therapy. Immunotherapies have recently shown remarkable results in the treatment of cancer patients. However, there are still many questions that remain to be solved in regards to more effective therapies, such as the tumor heterogeneous profile, tumor microenvironment, and tumor survival epigenetic and genetic pathways, all of which make patients resistant to the presently available treatments for cancer. This book demonstrates different approaches to overcome the challenges faced by immunotherapies due to suppressive tumor microenvironments. This book includes 18 papers that can be divided into three chapters: 1. novel immunotherapies; 2. targeting tumor microenvironment and novel approaches; 3. targeting tumors and tumor microenvironment in different types of cancer

Regulation of chondroprogenitor cell gene expression and migration

Cartilage, which lines joint surfaces to allow near-frictionless movement, lacks the ability to adequately repair itself and there are currently no effective, disease-modifying drugs to halt or repair the damage. Development of powerful in vitro models to investigate gene expression changes during osteoarthritis and chondrogenesis is key in understanding how the disease develops and how cartilage might attempt to repair itself. In this thesis, an enhanced model of chondrogenesis of the murine ATDC5 chondroprogenitor cell line was developed with cells cultured in micromass. Results revealed not only an increase in chondrogenesis markers, but markers of growth plate differentiation, including type X collagen, were either restricted or repressed, whilst expression of genes rich in articular cartilage were upregulated. This suggests that the enhanced ATDC5 model is more reminiscent of articular cartilage, making this model suitable for investigations into osteoarthritis - a disease of articular cartilage. The role of WNT5A signalling was then investigated (which is up-regulated in osteoarthritic cartilage) in a disease-like context by stimulating cultures with cytokines. Microarray analysis unearthed interesting and novel results, including a decrease in WNT5A signalling and in expression of members of the CCN family. Pathway analysis allowed further exploration of the interrelationship between cytokine and WNT signalling. Some changes in gene expression were reminiscent of those observed previously in in vivo models of early osteoarthritis. Finally, migration studies revealed that non-induced, undifferentiated ATDC5 cells have a migratory phenotype reminiscent of chondroprogenitor cells, which have the capacity to migrate to sites of cartilage injury in vivo. A novel model of cartilage invasion was also developed, with results suggesting WNT5A may be a potential inducer of chondroprogenitor invasion. Together, this thesis shows that the ATDC5 model is a good model for investigating articular cartilage both in a physiological and pathological setting