Reflux of gastroduodenal contents and consequent inflammatory responses are associated with the development of Barrett's oesophagus (BO) and the promotion of oesophageal adenocarcinoma (OAC). Deregulation of inflammatory processes is a hallmark of oesophageal cancer. In this study, we aimed to investigate (i) the transcriptional responses to deoxycholic acid (DCA) in cell lines representative of either end of the oesophageal cancer sequence, (ii) the expression of DCA-regulated genes in data charting oesophageal carcinogenesis and (iii) the impact of these genes on oesophageal inflammatory signalling. Gene expression microarrays were utilized to demonstrate differential transcriptional responses between squamous (HET-1A) and adenomatous (SKGT4) cell lines exposed to DCA. Differential basal and DCA-inducible expression of cytokines such as interleukin (IL) 8 was observed between both cell types. A cohort of DCA-regulated genes specific to each cell type was identified in microarray experimentation and subsequently validated. Cell type-specific genes included TRB3, CXCL14, GDF15 and LIF in HET-1A cells, with COX2-, ESM1-, URHF1- and IL1alpha-and IL1beta-specific expression in SKGT4 cells. Over 30% of the genes altered in BO and OAC were shown to be regulated by DCA utilizing an integrative genomic approach. One such gene, tribbles-homology-3 (TRB3) was induced specifically in HET-1A cells, absent in SKGT4 cells and decreased in BO samples in silico and in vivo. Inhibition and re-introduction of TRB3 in HET-1A and SKGT4 cells, respectively, demonstrated the ability of TRB3 to regulate inflammatory signalling through nuclear factor-kappaB. This study identifies mechanisms through which bile acids such as DCA, in conjunction with the loss of key signalling molecules, could regulate oesophageal metaplasticity.

Background & AimsEffective therapeutic approaches are urgently required to tackle the alarmingly poor survival outcomes in esophageal adenocarcinoma (EAC) patients. EAC originates from within the intestinal-type metaplasia, Barrett’s esophagus, a condition arising on a background of gastroesophageal reflux disease and associated inflammation.MethodsThis study used a druggable genome small interfering RNA (siRNA) screening library of 6022 siRNAs in conjunction with bioinformatics platforms, genomic studies of EAC tissues, somatic variation data of EAC from The Cancer Genome Atlas data of EAC, and pathologic and functional studies to define novel EAC-associated, and targetable, immune factors.ResultsBy using a druggable genome library we defined genes that sustain EAC cell growth, which included an unexpected immunologic signature. Integrating Cancer Genome Atlas data with druggable siRNA targets showed a striking concordance and an EAC-specific gene amplification event associated with 7 druggable targets co-encoded at Chr6p21.1. Over-representation of immune pathway–associated genes supporting cell growth included leukemia inhibitory factor, complement component 1, q subcomponent A chain (C1QA), and triggering receptor expressed on myeloid cells 2 (TREM2), which were validated further as targets sharing downstream signaling pathways through genomic and pathologic studies. Finally, targeting the triggering receptor expressed on myeloid cells 2-, C1q-, and leukemia inhibitory factor–activated signaling pathways (TYROBP–spleen tyrosine kinase and JAK-STAT3) with spleen tyrosine kinase and Janus-activated kinase inhibitor fostamatinib R788 triggered EAC cell death, growth arrest, and reduced tumor burden in NOD scid gamma mice.ConclusionsThese data highlight a subset of genes co-identified through siRNA targeting and genomic studies of expression and somatic variation, specifically highlighting the contribution that immune-related factors play in support of EAC development and suggesting their suitability as targets in the treatment of EAC.

Barrett’s oesophagus (BO), an intestinal-type metaplasia (IM), typically arising in conjunction with gastro-oesophageal reflux disease, is a prominent risk factor for the development of oesophageal adenocarcinoma (OAC). The molecular similarities between IM and normal intestinal tissues are ill-defined. Consequently, the contribution of intestine-enriched factors expressed within BO to oncogenesis is unclear. Herein, using transcriptomics we define the intestine-enriched genes expressed in meta-profiles of BO and OAC. Interestingly, 77% of the genes differentially expressed in a meta-profile of BO were similarly expressed in intestinal tissues. Furthermore, 85% of this intestine-like signature was maintained upon transition to OAC. Gene networking analysis of transcription factors within this signature revealed a network centred upon NR5A2, GATA6 and FOXA2, whose over-expression was determined in a cohort of BO and OAC patients. Simulated acid reflux was observed to induce the expression of both NR5A2 and GATA6. Using siRNA-mediated silencing and an NR5A2 antagonist we demonstrate that NR5A2-mediated cancer cell survival is facilitated through augmentation of GATA6 and anti-apoptotic factor BCL-XL levels. Abrogation of NR5A2-GATA6 expression in conjunction with BCL-XL co-silencing resulted in synergistically increased sensitivity to chemotherapeutics and photo-dynamic therapeutics. These findings characterize the intestine-like signature associated with IM which may have important consequences to adenocarcinogenesis.