The integrative role of Glycogen Synthase Kinase 3B (GSK3B) in adhesion-originating signals human oesophageal squamous cell carcinoma cells

A dissertation submitted to the Faculty of Science, University of the Witwatersrand in fulfilment of the requirements for the degree of Masters of Science. September 2017.The serine-threonine kinase glycogen synthase kinase 3β (GSK3β), has, in recent years, become established as hub for a myriad of intracellular signalling pathways. Many of these pathways have been implicated in cell cycle progression and proliferation in a multitude of carcinomas. GSK3β is ubiquitously expressed and considered to be constitutively active, and phosphorylation at the N-terminus serine 9 residue results in the inhibition. Interestingly, another prominent phosphorylation at tyrosine 216 in the activation loop has been reported to enhance GSK3β activity 200-fold. Its’ role in human oesophageal squamous cell carcinoma (HOSCC) migration, however is not well characterised. This study established that both active and inactive GSK3β are present in high abundance in HOSCC cells at rest. In order to characterise the influence of GSK3β on the migratory phenotype of HOSCC, focal adhesion kinase (FAK), a focal adhesion-associated protein known to be increasingly activated during cell migration (FAK(Tyr397)) was used as a marker for cell migration. The abundance of active GSK3β (pGSK3β(Tyr216)) was found to fluctuate during cell migration into a wound over 24 hours. Further investigation via the abrogation of GSK3β revealed that the observed variation was not a result of migration. Instead active GSK3β was found to differentially influence the migratory response observed in HOSCC cells by either 1.) promoting laemellipod extension and cell migration or 2.) partially-abrogate these processes. These findings however, did not produce the expected biochemical alterations with respect to the abundance of pFAK(Tyr397). Moreover, the effect of GSK3β-inhibition on HOSCC was shown to be dependent on the order in which wound initiation and GSK3β occurred, as abrogation of GSK3β prior to wound initiation was seen to either 1.) simply sustain the changes in the migratory response or 2.) produce little variation in the migratory response, suggesting the existence of “rescue-signalling”. These influences thus present GSK3β as a key regulator in HOSCC migration. Additionally culturing HOSCC cells on either collagen I of fibronectin, presented general decreases in the abundance of activated FAK, suggesting that varying suggesting that no one ECM-component, but rather the cell surface receptor distribution has become more diverse. This diversity may be a contributing factor to the varied influence of active GSKβ on the migratory response observed in HOSCC.LG201