AbstractThere are a subset of cancers that since their initial discovery and early efforts to develop treatments, still have a poor prognosis. These include Small cell lung cancer, Neuroblastoma and Glioblastoma and together they contribute to a large percentage of human cancer deaths. Efforts now focus on identifying novel targets that drive the initiation and development of these cancers to advance therapeutics in an advance towards personalised cancer therapies.
Aberrant post-transcriptional gene regulation has been implicated in numerous diseases including Neurodegeneration and Cancer. The family of Hu proteins consists of the neuronal HuB, HuC and HuD and ubiquitously expressed HuR. They are proto-type RNA-binding proteins functioning in all aspects of RNA processing including RNA stability, alternative splicing, polyadenylation, localisation and nuclear export. Hu proteins that are ectopically expressed or overexpressed in Small cell lung cancer (SCLC), Neuroblastoma and Glioblastoma have been linked to tumour progression.
Little research has been done to analyse how Hu proteins contribute to the development and progression in the described cancers. Silencing Hu proteins using siRNA interference provides an opportunity to analyse the effect of decreased Hu expression on cellular properties and the change in post-transcriptional regulation of target RNA that may play a role tumour formation and progression.
Effects of cell migration and cell viability were assessed in vitro and revealed HuB and HuC proteins to be key regulators of these processes. A decrease of HuB gene expression by RNA interference resulted in an increase in migration, in Glioblastoma cells U87-MG and the Neuroblastoma cell lines SH-SY5Y and SK-N-AS. Whilst a decrease in HuB and HuC gene expression showed an increase in viability and migration of the Neuroblastoma cells SH-SY5Y and Glioblastoma cells U87-MG suggesting these proteins act to control these factors in the cancers.
Individual and combined Hu gene knockdowns with siRNA also revealed regulatory and compensatory interactions of the Hu family members. HuR protein was found to positively regulate the expression of HuC in all cell models. In SH-SY5Y Neuroblastoma cells and U87-MG Glioblastoma cells, additional similarities were observed. HuR protein positively regulated HuD mRNA and HuC negatively regulated HuR mRNA. The HuR and HuC interactions suggests they can regulate each other’s protein levels ensuring an abundance of each protein in cells.
Molecular screening of a set of mRNA targets that have been described to contribute to the development of each cancer revealed many changes. From the array of targets that changed after knockdowns, genes with a high expression fold-change and influenced by more than one Hu protein were chosen to confirm the regulation by Hu proteins. Many of these targets were identified as members of the MAPK signalling pathway.
Further analysis including a complete knockout of the Hu proteins is needed to confirm the role of Hu proteins in regulating members of the MAPK signalling pathway and how the knockout would affect upstream and downstream targets of this pathway.
|Date of Award||Jul 2019|
|Supervisor||Irmgard Haussmann (Supervisor), Christopher Mee (Supervisor) & Helen Maddock (Supervisor)|