Bufe 2017 TRACT PhD project abstract

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Metabolic profiles in normal, dysplastic and cancerous oral cells.

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Bufe A (2017)

Event: TRACT

To this day, cancer is still among the leading causes of death worldwide and predicted to catch up with heart diseases, which holds the first position for now, very soon 1. The treatment is complex and in many cases ineffective. Especially for patients diagnosed with oesophageal cancer (OC) the odds are bad, which could be told by a 5-year survival rate of only about 18%. Furthermore, the number of new cases and deaths did not markedly decrease over the last years for this type of cancer 2. One reason for this is that up to this point no clinically useful molecular prognostic biomarkers exist and treatment options are very poor 3. Therefore, it is strongly required to explore new ways and tools to carry on the fight against OC and cancerous diseases in general. As it was found out during the last years that cells undergo a distinct metabolic remodeling when turning into tumour cells, the investigation of this process might be highly relevant for future cancer research 4. Pilot studies performed in the Queen’s University of Belfast (QUB) using a pathway based approach to identify determinants of drug resistance in oral and oesophageal cancer (OOC) have already identified the glycolytic pathway as a potential targetable pathway. With this, particularly mitochondria, which are the main energy producers of the cell and are involved in multiple regulatory pathways, may play key factors in the carcinogenesis and therefore should be focused over the course of this work.

The stated PhD project will be conducted in the frame of the European Union's program “TRACT”, funded by a Marie Skłodowska-Curie fellowship. With the company Oroboros Instruments as a partner, it will aim at the examination of metabolic transformation mechanisms in OC with the attempt of identifying new drug targets for future therapeutic development and new diagnostic strategies.

The work will further characterize the bioenergetic and metabolic properties of normal, metaplastic, dysplastic and cancerous oesophageal cells and identify differences between these stages of carcinogenesis. The study will include comparison of oxygen consumption, extracellular acidity and metabolic flux under normoxic and hypoxic conditions. The measurements will be conducted using an Oroboros Oxygraph 2k (O2k)-Fluorometer enabling the screening of real-time bioenergetics and metabolism of cells by the combination of high resolution respirometry (HRR) with fluorometry. By this, not only the cellular respiration (O2 consumption) of cells or mitochondria can be analyzed, but also multiple other metabolic parameters including the mitochondrial membrane potential, ATP production (indirectly via the concentration of free Mg2+), Ca2+ production and reactive oxygen (ROS) production (via H2O2 concentration) 5. These parameters are often altered in cancerous cells and might therefore be interesting for diagnostic and therapeutic interventions. To name an example, the production of ROS in mitochondria is known to be an essential component of multiple cellular pathways; however, an overload of oxidative stress can also cause genetic and functional damage, probably leading to degenerative diseases or cancer 6. By measuring the oxidative stress as a signal of hydrogen peroxide production and decreasing or increasing it by adding specific chemicals, it could be further evaluated how significant the effects of ROS production might be in the development of OC. Over the course of a 9-month secondment at the Trinity College of Dublin (TCD) under the supervision of Prof. Richard Porter, metabolic flux will be measured additionally through glycolysis, pentose phosphate pathway and glutaminolysis using 2H/13C NMR to complement the respiratory and fluorometric measurements conducted with the O2k at Oroboros Instruments. The stated experiments and investigations will be performed with cell culture models of oesophageal adenocarcinoma as well as human tissue biopsies of different stages of cancer -namely intestinal metaplasia, dysplasia, adenocarcinoma and non-cancerous tissue as control. For the conduction of the experiments, an ethics committee vote is obtained. To enable the repeated measurement of the samples, parts of all received samples are intended to be cryopreserved. Anyway, the feasibility of the cryopreservation of these tissues remains to be tested and will be proven by simultaneous comparative measurements of the cellular functions and metabolism of cryopreserved as well as fresh tissues with the O2k. By comparing the different types of cancerous tissue with the cell culture models of adenocarcinoma, the representativeness and reliability of the utilized cell models regarding their metabolic properties will be tested. With the inclusion of human tissues, the relevance and applicability of this study for the clinic is particularly ensured.

On the whole, the main aim of this thesis is the identification of differences in the metabolic profiles of the examined states of cancer and, in hand with this, the metabolic transformation mechanisms taking place in OC. In so doing a basis of differential novel drug targets will be established and the progression of means to enhance the chemotherapeutic sensitivity of cancer cells identified.

References

  1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide, IARC CancerBase, No. 11 (2013).
  2. Howlader N, Noone AM, Krapcho M, Miller D, Bishop K, Altekruse SF, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA. SEER Cancer Statistics Review, 1975-2013, National Cancer Institute (2016).
  3. McCormick Matthews LH, Noble F, Tod J, et al. Systematic review and meta-analysis of immunohistochemical prognostic biomarkers in resected oesophageal adenocarcinoma, British Journal of Cancer, 113(1):107-118 (2015), doi:10.1038/bjc.2015.179.
  4. Smolková K, Plecitá-Hlavatá L, Bellance N, Benard G, Rossignol R, Ježek P. Waves of gene regulation suppress and then restore oxidative phosphorylation in cancer cells, The International Journal of Biochemistry & Cell Biology, 43(7):950-968 (2011), ISSN 1357-2725, doi: 10.1016/j.biocel.2010.05.003.
  5. Fasching M, Gradl P and Gnaiger E. The O2k-Fluo LED2-Module, Mitochondrial Physiology Network, 17.05(08):1-6 (2015).
  6. Camara AKS, Lesnefsky EJ, Stowe DF. Potential Therapeutic Benefits of Strategies Directed to Mitochondria, Antioxidants & Redox Signaling, 13(3):279-347 (2010), doi:10.1089/ars.2009.2788.


Bioblast editor: Bufe A O2k-Network Lab: AT Innsbruck Oroboros


Labels: MiParea: Respiration  Pathology: Cancer 

Organism: Human  Tissue;cell: Other cell lines  Preparation: Intact cells, Permeabilized cells, Permeabilized tissue 



HRR: Oxygraph-2k, O2k-Fluorometer