Oesophageal adenocarcinoma (OAC) is the 14th most common cancer, accounting for 5% of cancer mortalities in the UK. The genomic landscape of OAC is driven by structural variations and is characterised by high mutation burden. Although recurrent mutations are observed in only few genes, we have identified TRPA1 (Transient Receptor Potential Ankyrin 1) as a novel driver gene in OAC with recurrent mutations in a sequenced cohort of 551 OAC patients. TRPA1 is a ligand-gated, Calcium (Ca2+) -permeable membrane protein, which has recently been implicated in breast and prostate cancers. This doctoral thesis aims to uncover the tumour-promoting effects of TRPA1 in OAC, by using a multi-disciplinary approach including bioinformatics, molecular modelling, biophysics, network modelling, and molecular biology techniques.

Work presented in this thesis identifies missense point mutations in TRPA1. The location of these mutations along the protein, reveals a mutation cluster in the intracellular N-terminal region, namely the Ankyrin Repeat Domain (ARD). The cryo-EM structure of TRPA1 is partially resolved, and the first 445 aminoacids spanning the ARD are missing from the crystallised structure. Through homology modelling, a full-length model of TRPA1 is constructed and the protein’s dynamics are studied by Molecular Dynamics (MD) simulations. MD analyses in coarse- grain (CG) resolution reveal a flexible hinging of the otherwise rigid ARD. OAC point mutations are located on the predicted flexible loop, and I hypothesize that these mutations alter protein dynamics and flexibility. CG-MD simulations of the full-length protein embedded in a lipid bilayer show that TRPA1 mutations neither enhance nor hinder the flexibility observed in the ARD.

By applying in silico methods, ten OAC point mutations are selected for downstream experimental validation, and their effect on Ca2+ flow is measured using Fura-2 Ca2+ imaging in 2D cell lines. Exogenous vectors containing mutant and wild-type TRPA1 are introduced to cells via lentiviral transduction. Missense mutations E179K, K186N, Q204K, K390N and H1018R reduce Ca2+ flow compared to WT, while L290P in the ARD, causes complete loss of Ca2+ entry following stimulation with Cinnamaldehyde, a TRPA1-specific agonist. OAC missense mutations do not cause significant changes in cell growth and proliferation, as measured by the Incucyte® platform.

A network modelling approach is then employed to explore the role of TRPA1 in cellular Ca2+ signalling dynamics, and to simulate TRPA1 loss-of-function and gain-of-function scenarios in a cancer cell. First, a static disease map is built by performing a literature review on known Ca2+ effectors in OAC, using CellDesigner. The static network is then converted to an executable, dynamic network, where changes in the cell’s Ca2+ signalling apparatus are explored in silico, using the BioModel Analyser (BMA) software. This data, in combination with Gene Set Enrichment Analysis (GSEA) from OAC patient data, identify hypoxia and pro-apoptotic pathways to be dysregulated following changes in TRPA1 expression.

This doctoral thesis reveals a multi-scale approach towards better understanding the complex processes by which TRPA1 can promote oncogenic processes in OAC.