MSACL 2019 EU Abstract

Investigating the Relationship between TRIM44 and P53 Metabolic Pathways in Human Colon Cancer Cells in a 3D Model, Using PTR-ToF-MS

Katerina-Vanessa Savva (1), Ilaria Belluomo (1), Anke M. Nijhuis (1), Hector Keun (1), George B. Hanna (1), Christopher J. Peters (1).
1) Department of Surgery and Cancer, Imperial College London, London, United Kingdom

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Katerina-Vanessa Savva (Presenter) PhD, Imperial College London

Presenter Bio: Katerina Vanessa Savva is a first year PhD student at Imperial College London, department of Surgery and Cancer. Upon completion of her BSc in Biomedical Sciences from the University of Sheffield (2017), she pursued an MRes in Biomedical Research at Imperial Collage London (2018) where she started working with mass spectrometry applied to breath research. Her PhD research project focuses on the development of a biomarker assessment toolkit which will mediate the successful translation of biomarkers from the ‘bench’ to the clinic. Additional projects of Katerina’s PhD involve i) characterisation of the relationship between TRIM44 and p53 metabolic pathways using proton transfer reaction time-of-flight mass spectrometry and ii) Identification of Volatile Organic Compounds associated with bacterial overgrowth in post gastrointestinal surgery patients.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Background: Colon cancer is the fourth most common cancer in the UK resulting in approximately 16,000 deaths per year (Cancer Research UK, 2019). Biomarkers capable of risk stratification or leading to targeted therapy are therefore necessary to promote prompt patient diagnosis and treatment. This raises the need to understand cancer related metabolic pathways, and subsequently identify diagnostic and prognostic biomarkers. Overexpression of the gene TRIM44 has been related with carcinogenesis in a variety of cell lines and biospecimens, emphasising its importance as a potential cancer biomarker. However, TRIM44 mechanism of action is not yet elucidated. At the protein level, TRIM44 is structurally similar to TRIM29, which was previously shown to interact with p53, a crucial cell cycle regulator, by mediating its nuclear export. Taking into consideration the structural similarity between TRIM44 and TRIM29, this study hypothesised that TRIM44 mediates its carcinogenic effects is via p53-related pathway.

Objectives: This project aims to i) identify volatile metabolites linked with p53 and TRIM44 cellular mutations and ii) to study the association between p53 and TRIM44 metabolic pathways in Human colon cancer cells (HCT116).

Method: To identify p53 generated metabolites in cell headspace, HCT116 cells, stably expressing differential levels of p53 (-/- p53, -/+ p53 and +/+ p53) are utilised. Cells are seeded on 3D collagen hydrogels which resemble the native environment of cells. High-throughput, online measurements of Volatile Organic Compounds (VOCs) are obtained from cell headspace using PTR-ToF-MS. Different cell concentrations and incubation times (0h, 8h, 16h, 24h) are used to assess optimal condition for VOC assessment. The same protocol is applied in HCT116 with differential expression of p53 (-/- p53, -/+ p53 and +/+ p53), upon knockdown of TRIM44. Metabolite identification upon TRIM44 knockdown is compared to wt TRIM44 HCT116 cell lines containing differential p53 expression levels.

Results: A novel and highly sensitive method, not requiring sample preparation, is developed and optimised to assess VOCs released in cell headspace, using PTR-ToF-MS. In particular, this project characterises metabolites linked with p53 and TRIM44 mutations in HCT116 cells. Subsequent result analysis and VOC comparison would define the relationship between p53 and TRIM44 metabolic pathways.

Impact: This study increases the understanding of TRIM44 mechanism of action in human colon cancer cells. Identification of volatile metabolites linked with p53 and TRIM44 mutations is a step closer to reaching the ultimate goal, the non-invasive characterisation of these mutations via VOCs in exhaled breath. Moreover, as TRIM44 has been shown to be important in a number of other cancers this knowledge will also benefit the wider research community.