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Title: An investigation into the expression and the role of Inhibitory Factor 1 in pancreatic acinar cells and pancreatic cancer
Author: Tanton, H. E.
ISNI:       0000 0004 7428 4392
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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When normal mitochondrial respiration is impaired (e.g. in ischemic conditions), the mitochondrial membrane potential usually becomes compromised, in which case it becomes more energetically favourable for the F1F0-ATP synthase to work in reverse as an ATP-hydrolysing proton pump. This can help to sustain the mitochondrial membrane potential, or at least slow the rate of its decline by pumping protons from the mitochondrial matrix. However, in these conditions, the reverse mode of F1F0-ATP synthase consumes a large amount of ATP which can subsequently lead to cell death. During this period of impaired respiration, the cell is temporarily able to use ATP produced by glycolysis as its energy source. The role of IF1 is to bind to F1F0-ATP synthase and inhibit its ATPase activity, thereby protecting the cells ATP stores from depletion, at the expense of accelerated loss of mitochondrial membrane potential (ΔΨm). The level of IF1 expression varies dramatically in different tissues and even between cell types of the same tissue. IF1 is overexpressed in many different carcinomas compared to the respective normal tissue, and confers an advantageous metabolic phenotype to cancer cells. High levels of IF1 have also been linked to increased glycolysis, resistance to cell death, increased migration, invasion and proliferation. In addition, high levels of IF1 have been shown to predict poor prognosis and correlate with advanced disease in many cancers, demonstrating its potential use as a marker of disease progression and as a therapeutic target. However, neither the expression nor role of IF1 has been studied in the normal pancreas or in pancreatic cancer. In this study, immunofluorescent and Western blot analysis was used to determine the expression of IF1 in normal pancreas and pancreatic adenocarcinoma (PDAC) cell lines; IF1 expression is low in normal pancreas in comparison to normal heart tissue which demonstrates high IF1 expression. We found that PDAC cell lines have a higher IF1 content and IF1/ATP synthase ratio than pancreatic acinar cells. In addition, we reveal that acinar cells and PDAC cells preferentially express different IF1 isoforms with the molecular weights 12kDa and 8kDa respectively. Using confocal microscopy, we demonstrated that the level of IF1 expression in PDAC cells determines the dissipation rate of the mitochondrial membrane potential in a model of chemical hypoxia. We also showed with this model that the level of IF1 expression determines cellular ATP consumption. Using Seahorse XF analysis, we found that IF1 up-regulates glycolysis in PDAC cells, which may help to confer resistance to hypoxia and increase both proliferation and invasion. Indeed, we demonstrated that IF1 knockdown and knockout in the PANC1 cell lines, and IF1 knockdown in the MIA PaCa-2 cell line, decreases their migration, invasion and proliferation, which suggests that IF1 may play an important role in PDAC growth and metastasis. However, using an orthotopic mouse model of PDAC, we did not find any difference in tumour growth between mice that were injected with wild-type PANC-1 cells, and those that were injected with IF1-/- PANC1 cells. There were also no resolvable differences in fibrosis, apoptosis and proliferation between the experimental groups. We found that human PDAC patients express higher levels of IF1 in PanIN and cancerous cells than in normal pancreatic acinar and ductal cells. PDAC tissue micro arrays were used to examine IF1 and ATP synthase expression in ~96 patients, and to explore whether any correlations exist between the expression of these proteins and patient survival. We did not find any significant correlations between IF1 or ATP synthase expression with prognosis, but this experiment did reveal that IF1 expression is significantly higher in PanIN than pancreatic acinar cells. This result suggests that up-regulation of IF1 may be important for early pancreatic carcinogenesis.
Supervisor: Tepikin, Alexei ; Costello-Goldring, Eithne ; Schmid, Michael Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral