Author: Lisa Pogson
Pogson, Lisa, 2021 Elucidating the role of dipeptidyl peptidase 4 in cancer metabolism and biology using in vitro and in vivo models, Flinders University, College of Science and Engineering
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Diabetes and cancer are two diseases with significant impact on worldwide health that share many mutual risk factors and biological links. While both disease states involve significant metabolic change as well as activation of inflammatory and stress-related pathways, a causal relationship between diabetes and cancer is yet to be identified. Patients with diabetes have a significantly higher risk of developing certain cancer types including colorectal cancer, the second leading cause of cancer related deaths worldwide with about 1 million cases and >500,000 deaths annually. Furthermore, an increase in colorectal cancer incidence is associated with western diet and lifestyle, factors that are also linked with increased insulin resistance and predisposition to type 2 diabetes mellitus (T2DM), such as high calorie intake, low physical activity and excess abdominal fat. Recent observational studies suggest that some medications used to treat hyperglycaemia and T2DM are associated with a reduced risk of cancer. Given this association, there is a growing interest in determining the impact of T2DM treatment targets on tumorigenesis.
Sitagliptin is a selective dipeptidyl peptidase‐4 (DPP4) inhibitor also used for the treatment of T2DM. The insulinotropic action of sitagliptin involves the increase of nutrient‐stimulated insulin release through inhibition of DPP4 enzymatic degradation of the active form of incretins, glucose-dependent insulinotropic peptide (GIP) and glucagon‐like peptide‐1 (GLP‐1). Sitagliptin is one of the most readily available DPP4 inhibitors in Australia, in the 2019/20 financial year 1.5 million pharmaceutical scripts for sitagliptin were claimed in Australia amassing a total cost of $82 million dollars. However, in addition to its insulinotropic action, the enzymatic activity of DPP4 is shown to be involved in a diverse range of biological processes associated with neoplastic transformation, including cell differentiation and adhesion, immune modulation and apoptosis. This raises some well warranted concerns within the research and medical community on whether DPP4 inhibition will have a positive or negative effect on the initiation and progression of different cancers, particularly colorectal cancer. The overall aim of this thesis was to determine the role DPP4 inhibitors play in the control of the metabolic needs of cancer cells and establish a relationship between DPP4 and colorectal cancer development and progression.
In chapter two sitagliptin was used to examine the possible effects of DPP4 inhibition on the regulation of cellular metabolism and the AMP-activated protein kinase (AMPK) signalling pathway in two lymphocyte cell lines. The actions of sitagliptin were compared to metformin a known AMPK regulator and front line T2DM treatment associated with a reduced cancer burden in diabetic patients. In vitro analysis of mitochondrial respiration and glycolysis demonstrated that sitagliptin alters the bioenergetic profile of both T lymphocyte cells and human acute monocytic leukemia cells, reducing both oxidative phosphorylation and glycolysis in a similar fashion to metformin. Additionally, metabolic reprogramming by sitagliptin was associated with increased AMPK and activated AMPK presence, indicating that DPP4 imparts control over cellular metabolism through AMPK associated actions.
In chapter three a chemically induced colon carcinogenesis model using azoxymethane (AOM) was established in DPP4 genetic knockout mice (Dpp4-/-) on a the C57Bl/6 background. In this work the closely related DPP4 protein family member fibroblast activation protein (FAP) was also targeted using the same model of carcinogenesis in FAP genetic knockout mice (Fap-/-). FAP shares a close sequence identity to DPP4 and is believed to arise from gene duplication due to its gene proximity and is identified as a potential pharmaceutical target in several types of cancers including colon carcinogenesis. Chapter three is the first study to utilise the AOM model of colorectal cancer in Dpp4-/- and Fap-/- mice and establishes that this model of colorectal cancer is suitable for use with these genetic knockouts. In chapter three a monitoring refinement was established that led to improvements in animal welfare by reducing the weight loss cut off point for euthanasia. This enabled identification of a subset of mice where AOM treatment led to severe liver damage allowing elimination of these mice earlier from the study. This modification ties into the refinement alternative of the three R’s principal of animal experimentation reducing animal distress and enhancing the welfare of experimental animals. Additionally, this study indicates that DPP4 and FAP ablation is protective against severe AOM driven liver injury and less mice in these groups were removed from the trial as they were less likely to reach the 10% weight loss cut-off for euthanasia after AOM treatment.
In chapter four the refined AOM model described in chapter three was used to examine the role of DPP4 and FAP in CRC progression. Chapter four provides further evidence that both DPP4 and FAP play a role in tumour pathogenesis. With exposure to AOM, Dpp4-/- mice develop a significantly increased number of adenomas and adenocarcinomas while both Dpp4-/- and Fap-/- mice have significantly increased tumour growth and penetrance. Dpp4-/- mice also show a significant reduction in total numbers of ACF which indicates that in the absence of DPP4 tumour progression is occurring at a faster rate. Dpp4-/- and Fap-/- mice have reduced plasma levels of CXCL10 indicating a reduced ability to recruit immune cells into the tumour site, additionally, Dpp4-/- mice have significantly reduced lymphocyte numbers indicating that both phenotypes have an impaired immune response which may be contributing to increased tumour growth. The results from this work confirm that both DPP4 and FAP are involved in CRC tumour development and highlights the need for further work to identify the mechanisms through which ablation of DPP4 and FAP increases tumorigenesis.
The significant original contribution of knowledge in this thesis is that it is the first study to demonstrate using the AOM chemically induced carcinogenesis model in Dpp4-/- and Fap-/- mice, that both DPP4 and FAP play a role in tumour incidence and development and that DPP4 plays a significant role in tumour prevalence. In contrast chapter two indicates that sitagliptin may provide a novel therapeutic approach for targeted metabolic reprogramming of cancer cells positioning DPP4 inhibitors as potential anti-cancer agents for some specific tumour types. These findings are in direct contradiction to recently published animal studies suggesting that DPP4 inhibitors either have no effect on tumour development or decrease overall tumour number. However, DPP4 has two domains, an enzyme active domain, which DPP4 inhibitors act upon, and another domain that is able to mediate protein-protein interactions that are involved in the cancer process. This paradox suggests that although inhibition of DPP4 enzyme activity may be protective against CRC development, DPP4 direct protein interactions may be involved in a much more significant way in control of colorectal cancer development and progression.
Keywords: Dipeptidyl Peptidase 4, DPP4, Fibroblast Activation Protein, FAP, Diabetes, Colon Cancer, Sitagliptin, Metformin
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
Completed: 2021
School: College of Science and Engineering
Supervisor: Professor Catherine Abbott