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Understanding the biology of reactive oxygen species and their link to prostate cancer
thesisposted on 27.02.2017, 00:07 by Harrison, Ian Paul
The family of NADPH oxidases exist solely to generate reactive oxygen species (ROS) such as superoxide (O2ˉ) and hydrogen peroxide (H₂O₂). ROS are important signalling molecules within the body and are essential to many physiological processes, such as cellular proliferation. However, if there is an imbalance between ROS production and metabolism oxidative stress can occur, potentially giving rise to many diseases including cancer. The exact subcellular location of ROS production is unclear, but a growing body of evidence suggests that it may be within the endosome that this ROS is being produced. Thus, this thesis examined the hypothesis that NOX2-NADPH oxidase-derived ROS is crucial in the pathogenesis of prostate cancer and that a key sub-cellular location for this ROS production is the endosome. Using a syngeneic, orthotopic model of mouse prostate cancer, we found in Chapter 3 that genetic deletion of NOX2 significantly inhibited prostate tumour growth whereas endothelial NOX4 overexpression had no effect on prostate tumour growth. In NOX2-/y tumours we also observed a significant inhibition in tumour angiogenesis and a significant increase in tumour-associated macrophages. Taken together, these data demonstrate that NOX2 is a critical mediator of prostate tumourigenesis through the modulation of the stroma surrounding the prostate gland. Interestingly, in analysing human prostate tissue we found that NOX2 expression is significantly elevated in primary prostate cancers compared with healthy prostate tissue and that NOX4 expression was only elevated in metastatic prostate cancers. These results showed for the first time that NOX2 is a critical component of primary prostate cancer, and provide a potential therapeutic target to treat prostate cancer. ROS are known to promote growth factor signalling such as VEGF. In Chapter 4, we sought to determine the subcellular location of ROS generation. Using our syngeneic, orthotopic mouse model of prostate cancer, we found that interventional treatment with a VEGF inhibitor significantly decreased tumour development. In endothelial cells treated with VEGF-A, we found that the early endosome marker EEA1 co-localised with NOX2 and the VEGFR2. Furthermore, we found that treatment with VEGF-A significantly increased NOX2-dependent ROS production within acidified endosomal compartments. Additionally, we found that VEGF-A-stimulated endothelial proliferation was also dependent on VEGFR2 endocytosis, endosomal acidification and NOX2 activity. These results shed further light on those obtained in Chapter 3, and provides a rationale for targeting endosomal NOX2 in prostate cancer. In Chapter 5, we pharmacologically inhibited NOX2 in the same model of prostate cancer as that used in Chapters 3 and 4. We found that interventional treatment with a combined oral and i.p administration of the NOX2 inhibitor/ ROS scavenger apocynin in a mouse with an established tumour significantly inhibited prostate tumour growth, again, as in Chapter 3, showing the critical importance of NOX2 in prostate tumourigenesis. However, pharmacological inhibition of NOX2 did not alter either angiogenesis or the immune response. This study further expands upon the results of Chapter 3 and provides a rationale for targeting NOX2 in vivo with pharmacological inhibitors. In summary, the findings of this thesis indicate that (1) NOX2-derived ROS plays a critical role in prostate tumourigenesis through the modulation of the prostate stroma and (2) the sub-cellular location of this NOX2-derived ROS is within the endosome. While future work is needed to fully elucidate the role of both NOX2 and the other NADPH oxidase isoforms in primary and metastatic prostate cancers, the findings within this thesis firmly establish NOX2 as a leading candidate for the origin of the excessive ROS production associated with prostate cancer. As such, we propose that endosomal NOX2 may represent a novel therapeutic target in the treatment of prostate cancer.