Selective autophagy receptors in pancreatic ductal adenocarcinoma
Thesis or Dissertation
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Pancreatic cancer accounted for 2.5% of all new cancers diagnosed worldwide in 2018, but remains disproportionally one of the most lethal cancers. Therefore, the development and validation of novel biomarkers (BMs), for the most prevalent form of the disease (pancreatic ductal adenocarcinoma (PDAC)), is key to develop more efficacious and kinder treatments. In particular, PDAC cells have high growth and protein production rates. A portion of these proteins misfold, which the cell normally controls, by using the proteasomal degradation system, but under stressful conditions, eg. chemotherapy or radiation, the proteasomes can be overwhelmed. Pharmacological interventions, such as proteasomal inhibitors (PI) (Velcade/Bortezomib), can increase this protein stress. Misfolded proteins nor degraded by the proteasome eventually aggregate which are then degraded by a selective type of autophagy called aggrephagy. It is hypothesised that aggrephagy is a resistance mechanism used by cells in the PDAC disease setting to adapt to protein stress. Certain PDAC tumours rely heavily on ATG4B-driven autophagy. Clinicians could combine inhibition of both, proteasomal and ATG4B-driven autophagic degradation of misfolded proteins, for treating PDAC. this could cause overwhelming protein stress in the tumour, while sparing normal tissues with physiological levels of protein production, thus providing a therapeutic window. This study aims to explore the hypothesis of an autophagy-inhibiting therapy designed to stop the aggrephagy resistance pathway, exploited through the dependence of aggrephagy on ubiquitin-binding selective autophagy receptors (SARs). After proteotoxic stress, the SARs bind to the aggregated proteins, targeting them for autophagic degradation and so resolving the stress. Importantly, this study contributes to establishing translational BMs for autophagy in the context of pancreatic ductal adenocarcinoma (PDAC), by making the SAR panel available to the community. The specific aims were first to establish four SARs, as a comparative panel of ubiquitin-binding BMs. While doing so in vitro, TAX1BP1 was shown to have the fastest degradation kinetics. Also, all SARs were found to accumulate more substantially in the Triton-X insoluble fraction, as measured by western blotting and the WES assay. Then I aimed to characterize how the ASRs interact with their cargo to cause the degradation of protein aggregates by aggrephagy. I discovered that the SARs all colocalised to the ubiquitin-positive aggresome, created by proteasomal inhibition. The inner core of the aggresomal structure was positive for the four SARs and ubiquitin, whereas the outer shell was stained by the PROTEOSTAT dye, for misfolded and aggregated protein. Also, the catalytically dead ATG4B-C74A localised to the aggresome but did not noticeably alter it. The BMs were validated, by inducibly overexpressing the dominant negative and catalytically dead ATG4B-C74A. By using ATG4B-C74A, I inhibited human PDAC autophagic flux, proven through the accumulation of: p62, TAX1BP1, NDP52, and LC3B. The fourth aim was to explore the combination of proteotoxic insults, and discover if it can provide a therapeutic window in vitro and in vivo, by using ATG4B-C74A expressing cells and Velcade. While addressing this aim, the combined treatment did not lead to enhanced tumour growth inhibition. However, the combined proteotoxic stress visualized in PDAC tumours, may potentially be predicted by the increase in p62+ aggresomes/aggregates as a pharmacodynamic (PD) immunohistochemistry (IHC) biomarker (BM).
Cell Death and Immunity
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Institute of Cancer Research (University Of London)