Neuroblastoma (NB) is the most common malignant solid tumour diagnosed in infants, accounting for ~15% of all childhood cancer-related deaths. Current patient risk stratification criteria are heavily reliant on the presence of a MYCN amplification, albeit only accounting for ~25% of patients. The inadequate prognostic risk stratification of patients results in children receiving either inefficient or excessive treatment with a myriad of severe lifelong side effects for survivors. Therefore, the identification and characterisation of novel biomarkers could not only identify new therapeutic targets but could also improve risk stratification and treatment planning. A comparative transcriptomic analysis of NB tumours (obtained from the chick embryo model) grown under normal oxygen tensions (normoxia, 21% O2) or hypoxia (1% O2), a model for aggressive NB tumours that correlates with poor patient prognosis, identified multiple significantly upregulated genes in aggressive (hypoxic) tumours specifically, with Zinc Finger DHHC-Type Palmitoyltransferase 23 (zDHHC23) and
Maestro Heat Like Repeat Family Member 6 (MROH6) exhibiting the best correlation with poor prognosis. This thesis sought to validate these expressed gene products as potential biomarkers in NB. I also investigated the molecular function of these two proteins under normoxic and hypoxic conditions, supplementing the currently limited available knowledge.
Commercially available antibodies for these two proteins were unsuccessful for use in either immunostaining, a procedure currently used as the ‘gold-standard’ of clinical biomarker screening, or for immunoblotting of endogenous protein, with all of the antibodies evaluated lacking specificity. Although targeted mass spectrometry assays were successfully developed, they lacked the sensitivity to detect endogenous proteins, likely due to low levels of protein expression. Therefore, I focused on the biochemical characterisation of these two proteins, cloning dual reporter HA-mCherry-protein and protein-mCherry-HA plasmids to facilitate immunoprecipitation of exogenously expressed protein and evaluation of sub-cellular localisation. I developed and optimised a HA-tag based immunoprecipitation protocol for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis to allow
identification of post-translational modifications (PTMs) and protein interaction networks. These experiments revealed extensive hypoxia-induced regulation of protein binding
partners, with ~70% of the interactome (from a total of 262 and 253 co-immunoprecipitated proteins for zDHHC23 and MROH6 respectively) changing as a function of O2 tension. GOterm analysis of these interactomes suggests that zDHHC23 is a component of several potentially important malignancy pathways, including cytoskeletal reorganisation and adhesion. Label free quantification analysis of MROH6 identifies high stoichiometric binding to Breast Cancer Anti-oestrogen Resistance protein 1 (BCAR1), inferring potential roles in telomere maintenance and genetic stability. Additionally, PTM analysis identified one and three phosphorylation sites on MROH6 and zDHHC23 respectively, with zDHHC23 S252 predicted to be regulated by Cyclin dependent kinases. Finally, I developed, to my
knowledge, the first reported click-chemistry based high-throughput LC-MS/MS pipeline for the unbiased identification of zDHHC23 palmitoylated substrates, concluding that the ‘palmitome’ is much more complex than currently understood and likely regulates localisation to membrane bound organelles and extracellular vesicles, as well as its established role in plasma membrane localisation.
Overall, using LC-MS/MS approaches, I explore and discuss how zDHHC23 and MROH6 overexpression may contribute to aggressive NB development and poor patient prognosis
