Functional characterisation of phosphodiesterase 4D7 in prostate cancer

3’,5’-cyclic adenosine monophosphate (cAMP) is the best studied intracellular second messenger. Adenylyl cyclase (AC) catalyses the synthesis of cAMP from ATP following the stimulation of a G protein coupled receptor (GPCR), and its degradation is catalysed by cAMP phosphodiesterases (PDEs) to allow cessation of signal. cAMP can act to bring about a multitude of varying and often opposing cellular responses, which depend on the stimulus received by the GPCR, the cell type, the cell cycle stage, and the complement of downstream effector molecules within that cell. The cAMP PDE subfamilies express multiple splice variants, which possess unique N-termini and non-redundant functional roles. By virtue of this, they are targeted to specific and discrete subcellular locations, where they may form highly specific interactions with scaffold proteins and other enzymes. Here, in these discrete locales, PDEs act to hydrolyse local cAMP, thereby underpinning the spatial and temporal compartmentalisation of cAMP gradients. This fine-tuned balance of synthesis and degradation is paramount for the dynamic cellular responses to extracellular stimuli, allowing differing signal transduction cascades to occur simultaneously in the crowded macromolecular environment of the cell. The compartmentalisation of cAMP signalling is, thus, essential for maintaining cellular homeostasis, and is subject to perturbation in various diseases, including prostate cancer (PC). Despite the wealth of literature implicating cAMP signalling in the progression of PC, little work has been done on the expression or function of PDE splice variant in this disease. Our group, in collaboration with Philips Research and the Prostate Cancer and Molecular Medicine (PCMM) group in the Netherlands, set out to investigate the changes in cAMP signalling during PC progression by studying the expression of cAMP PDE isoforms, with the aim of identifying a novel PC biomarker, as the current standard biomarker (PSA) is not disease-specific and leads to much over-diagnosis and over-treatment of otherwise non-life threatening prostate tumours. Interestingly, we found PDE4D7 to be dramatically downregulated as PC progresses from an androgen sensitive (AS) to an androgen insensitive (AI) state, and, indeed, this enzyme is showing promise as a novel, disease-specific PC biomarker. In this thesis, I report my efforts to characterise a function of PDE4D7 within prostate cancer. Firstly, I report the raising of a novel highly specific PDE4D7 antibody and describe the differential expression of this isoform, at the protein level, between AS and AI PC cell models. I present evidence to suggest that PDE4D7 mediates PC cell growth and migration, and that its loss may play a role in PC progression. I propose that an altered epigenome plays a role in the downregulation of PDE4D7 expression. I then report on the raising of a novel phospho-specific antibody and present evidence to show that PDE4D7 is regulated by PKA phosphorylation within its unique N-terminal region, and that this event confers negative regulation on enzyme activity. Finally, I describe my endeavours to elucidate a PDE4D7 protein-protein interaction that may help transduce PDE4D7-specific signals and maintain the enzymes cellular location