The role of PGRMC1 in cell biology and pancreatic cancer tumourigenesis and a novel mechanism for fibril formation from Acot7 protein

Two independent studies are presented in this thesis. The first study, primarily conducted under the supervision of Dr. M. Cahill, involves the investigation into the role of a MAPR protein, PGRMC1, in cancer cell biology using a human pancreatic cancer cell line in Chapters 1-6. Pancreatic cancer is an aggressive cancer associated with a high metastatic potential and a low survival rate of approximately five years. The prognosis for pancreatic cancer has not changed significantly for decades due to various factors including often being diagnosed at a late stage, and typically having very few early or distinguishable symptoms and unknown aetiology. The 5-year survival rate for pancreatic cancer in Australia is 8.7% and women have a lower risk (AIHW, July 2019). In Australia, 1 in 55 men and 1 in 74 women are diagnosed with pancreatic cancer by age 85. In 2016, there were close to 3000 deaths from pancreatic cancer in Australia and there are approximately 3000 new cases of pancreatic cancer diagnosed in Australia each year. Drug resistance to the current standard drug therapies for metastatic pancreatic cancer, such as gemcitabine, is high. To improve diagnosis and prognosis of pancreatic cancer, and to enhance the efficacy of the current therapies for pancreatic cancer, it is critical to identify protein targets that are altered in pancreatic cancer or are involved in the multiple mechanisms of cancer progression and metastasis. Identification of potential future targets of therapies for pancreatic cancer is critical. PGRMC1 presents an attractive target for anti-cancer therapies in pancreatic and other cancers. In this thesis, PGRMC1 phosphorylation mutant proteins were expressed in MIA PaCa-2 pancreatic cancer cells to investigate the effect of differential phosphorylation of PGRMC1 on cell proliferation, response to drugs, and on tumour growth in a mouse xenograft model. The expression of the different PGRMC1 proteins did not influence the cells response to several anti-cancer drugs, however did significantly affect tumour growth. Furthermore, interaction partners of PGRMC1 were identified, with and without treatment with the small molecule inhibitor of PGRMC1, AG-205. AG-205 has the potential to be used to target PGRMC1 in cancer and other diseases such as diabetes and neurodegenerative diseases including Alzheimer’s disease. However, the potential offtarget effect of AG-205 on other MAPR proteins requires investigation.
The second study, primarily conducted under the supervision of Prof. J. Forwood, involves investigation of amyloid fibril formation from ACOT7 protein in Chapter 7. ACOT7 is a member of the thioesterase family of enzymes that catalyse the hydrolysis of fatty acyl-CoAs to free fatty acids and CoA. Thioesterases have an important function in regulating the cellular levels of fatty acyl-CoA ligands for certain transcription factors and in aiding in maintaining lipid homeostasis. Dysregulation of lipid metabolism is associated with the development of neurodegenerative diseases. ACOT7 is highly expressed in the brain and has been described to play an important role in inflammation through regulation of arachidonic acid. Investigation into the role of Acot7 in inflammation in the Forwood laboratory lead to a novel finding that the structural instability of Acot7 may result in amyloid formation. We tested the propensity of Acot7 to form fibrils, and specifically, conditions that promoted or inhibited amyloid fibrils. The initial identification of Acot7 fibril formation by our research group was detected by the accumulation of protein complexes and “laddering” of SDS/boiling resistant complexes during SDS-PAGE. In collaboration with John Carver at ANU, the formation of fibrils was assessed by transmission electron microscopy, a Thioflavin T assay, circular dichroism and X-ray diffraction. The structure of Acot7 (PDB: 6VFY) is presented and a mechanism for fibril formation from Acot7, involving domain swapping, is proposed. This mechanism is based on the ability of the N-terminal and C-terminal hotdog domains to superimpose and switch domains, and represents a novel mechanism of amyloid formation.