Characterisation of the unfolded protein response in prostate cancer, and investigation of the ATF6 interactome using a modified mammalian expression system.

Prostate cancer (PCa) cells grow in an environment which is known to cause endoplasmic reticulum (ER) stress. This activates a process called the unfolded protein response (UPR) which PCa utilises in order to survive and adapt to the adverse environmental conditions. Using interdisciplinary approaches and tissue culture models representing different stages of PCa, this project investigated the link between the UPR and the key oncogenic driver of PCa, the androgen receptor. It has been observed that hormone responsive PCa utilises all three UPR arms in order to promote ER homeostasis and cell survival. The importance of the UPR during the progression of PCa to the castrate resistant stage was also assessed. Interestingly, UPR signalling was inactivated in castrate-resistant models of PCa, and the cells were instead dependent on the ER-associated degradation (ERAD) pathway in order to resolve the stress and survive. These findings have identified potential UPR vulnerabilities that can be targeted to prevent disease progression. Little is known about the structure and interaction partners of the UPR sensor ATF6, as protein expression has been shown to be problematic. To resolve this issue a stable tetracycline-inducible HEK293S GnTI(-) cell line for the expression of ATF6 was generated and mass spectrometry performed to characterise the ATF6 interactome. Sixty novel interaction partners of ATF6 were identified, most of which are associated with the cytoskeleton, such as Spectrin β-II and p195, which were validated by immunoblotting. It is hoped that the use of this modified expression system will provide an advantage in the process of expression, solubilisation and structure determination of ATF6 and of other membrane proteins.