PO-172 Role of lithocholic acid-induced cell signalling in oesophageal cancer progression model

Introduction A p53-checkpoint operates in the development of oesophageal adenocarcinoma (OAC) defined by prevalent TP53 gene mutations in patients with Barrett’s and high-grade dysplasia (HGD). The molecular genetic relationship between OAC and its precursor lesion, Barrett’s oesophagus, is poorly understood. The effective therapeutic targets should focus on early mutations of the disease, which become clonal in the later stage. Mutational context of some specific single nucleotide variants are common throughout the progression of the disease, suggesting exposure to common mutagens throughout the progression course. Material and methods TP53 null or truncated cell lines were generated by targeting exon 4 of TP53 gene using CRISPR/Cas9 technology. Cell cycle analysis and Annexin V5 stained cells were analysed by FACS after treatment with lithocholic acid (LCA) to recapitulate the reflux of bile acids in OAC. Changes in proteins levels and signalling pathways upon LCA exposure were identified using mass spectrometry and validated with western blotting. Immunohistochemistry (IHC) analysis of OAC and normal tissues was performed to check the expression of identified bile acid-induced genes in tumours. Results and discussions Oesophageal Barrett’s wt-p53 cell line CPA was used to define the effects of TP53 gene ablation on stress responses, cell survival, and mutation rates. FACS analysis revealed TP53 null cells sensitivity towards LCA via apoptotic pathway. Mass spectrometry analysis identified disrupted NDRG1 and TGFβ pathways. SMAD4 driver mutations have previously been reported exclusively to OAC, which provide a clear genetic boundary between OAC and HGD. Western blot analysis after LCA exposure showed increased levels of SMAD4-independent of p53 pathway. SMAD4 depletion in TP53 null cells stimulates cell-migration in the presence of inhibitory levels of LCA, corroborates the role of SMAD4 loss in metastasis. Loss of p53 also upregulates NDRG1 which increases migration in presence of LCA and was found to be a pro-invasive factor. IHC staining of OAC, lymph node and normal tissues showed heterogeneous expression of NDRG1 in tumour, mostly in membrane, suggesting its dynamic expansion on LCA exposure in vivo . Conclusion The recurrent reflux of bile acids in Barrett’s cells, especially LCA, could sensitise cells to harbour further mutations. LCA induces p53-independent upregulation of SMAD4 . Depletion of NDRG1 and SMAD4 in p53 null cells stimulates cell-migration in the presence of LCA, corroborating their role in metastasis.