Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.579410
Title: Control of cAMP signalling in the cellular migration of pancreatic ductal adenocarcinoma
Author: Burdyga, Alex
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2013
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
Pancreatic ductal adenocarcinoma (PDAC) is characterised by a very high mortality rate and is the 4th most common cause of cancer death (Siegel et al., 2012). The disease initially develops asymptomatically, and at the time of diagnosis patients usually have multiple metastases (Rhim et al., 2012). It would therefore be highly desirable to develop treatments which specifically impede the ability of PDAC cells to metastasise by interfering with the cellular processes responsible for efficient cellular migration. Intracellular signalling cascades, which utilise various signalling proteins, ultimately lead to the appropriate cell coordination and enable efficient cellular motility. One such signalling pathway that participates in the regulation of migration is controlled by the second messenger cyclic adenosine monophosphate (cAMP) (Howe, 2004). Several effectors of cAMP have been found which include protein kinase A (PKA) (Tasken & Aandahl, 2004), exchange factors activated by cAMP (EPAC) (Bos, 2006), and cyclic nucleotide-regulated cation channels (Biel, 2009). PKA has been intimately linked with several cellular processes which contribute towards cell motility. In most cases, the various specific effects of PKA signalling require selective targeting of the kinase into microdomains through interaction with A-kinase-anchoring proteins (AKAPs) (Pidoux & Tasken, 2010). Other cAMP effectors such as EPAC have defined roles in controlling various aspects of migration, such as cellular adhesion to the extracellular matrix (Bos, 2005). The effect of modulating cAMP signalling on the rate of migration has been investigated in several cancer types. Interestingly the results obtained were rather varied; both inhibition and stimulation of migration was observed (Chen et al., 2008; Baljinnyam et al., 2009; Grandoch et al., 2009; Shaikh et al., 2012). However, the effect of cAMP, and its effectors, on the rate of migration has not been investigated in PDAC; this was the main aim of this study. Classical cAMP elevating agents such as forskolin and 3-Isobutyl-1-methylxanthine (IBMX), as well as the cAMP analogue 8-Bromoadenosine 3’5’-cyclic monophosphate (8Br-cAMP), were found to inhibit migration of the PANC-1 cells. The role of cAMP signalling was further supported by the results of experiments utilising cAMP FRET sensors, which were imaged in live single cells. Further characterisation of cAMP effects in 4 other diverse PDAC cell lines yielded similar results, indicating that the mechanism of inhibition was common to all PDAC cell types tested. PANC-1 cell invasion was also inhibited by cAMP elevation. I went on to investigate events such as cell ruffling and focal adhesion assembly, which are processes closely associated with cellular motility. Dual transfection with a cAMP sensor and GFP tagged paxillin revealed a relationship between cAMP elevation and the loss of paxillin from focal adhesions, which was quickly reversible upon cAMP returning back to basal levels. Using a similar approach, peripheral cell ruffling was found to be inhibited by intracellular cAMP elevation. These results indicated that the inhibition of migration upon cAMP elevation was likely to occur as a result of immediate signalling events (and not due to cAMP-dependent changes in gene expression). The final part of the project concentrated on the individual contribution of the downstream effectors of cAMP, with particular emphasis on selective PKA and EPAC modulation. Utilising both PKA and EPAC sensors, I determined the appropriate concentrations of N6-benzoyl-cAMP (6Bnz) and 8-pCPT-2’OMe-cAMP (8pCPT) required to achieve selective PKA and EPAC activation respectively. Interestingly, I found that the two effectors had opposing actions; EPAC activation was found to induce migration, while PKA was found to suppress migration. Further investigation utilised a potent and selective PKA inhibitor peptide (PKI), which upon expression was found to prevent inhibition of ruffling, paxillin loss from focal adhesions, and inhibition of migration in response to cAMP elevation. Furthermore, it was found that suppression of basal PKA activity had a tendency to induce migration. I also utilised a cell permeable peptide (st-Ht31) which inhibits PKA interaction with AKAPs, thus effectively reducing its function by uncoupling the kinase from its specific signalling microdomains. The resulting effect was found to be a large potentiation of PANC-1 migration, which further highlighted the importance of PKA activity in the control of migration.
Supervisor: Tepikin, Alexey; Costello-Goldring, Eithne Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.579410  DOI: Not available
Keywords: QH301 Biology ; RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Share: