siRNA Library Screening Identifies a Druggable Immune-Signature Driving Esophageal Adenocarcinoma Cell Growth.

BACKGROUND & AIMS: Effective 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. METHODS: This 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. RESULTS: By 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 EAC 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. CONCLUSIONS: These 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.

The characterization of an intestine-like genomic signature maintained during Barrett's-associated adenocarcinogenesis reveals an NR5A2-mediated promotion of cancer cell survival.

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.

Golgi phosphoprotein 2 (GOLPH2) is a novel bile acid-responsive modulator of oesophageal cell migration and invasion.

BACKGROUND: The aetiology of Barrett's oesophagus (BO) and oesophageal cancer is poorly understood. We previously demonstrated that Golgi structure and function is altered in oesophageal cancer cells. A Golgi-associated protein, GOLPH2, was previously established as a tissue biomarker for BO. Cellular functions for GOLPH2 are currently unknown, therefore in this study we sought to investigate functional roles for this Golgi-associated protein in oesophageal disease. METHODS: Expression, intracellular localisation and secretion of GOLPH2 were identified by immunofluorescence, immunohistochemistry and western blot. GOLPH2 expression constructs and siRNA were used to identify cellular functions for GOLPH2. RESULTS: We demonstrate that the structure of the Golgi is fragmented and the intracellular localisation of GOLPH2 is altered in BO and oesophageal adenocarcinoma tissue. GOLPH2 is secreted by oesophageal cancer cells and GOLPH2 expression, cleavage and secretion facilitate cell migration and invasion. Furthermore, exposure of cells to DCA, a bile acid component of gastric refluxate and known tumour promoter for oesophageal cancer, causes disassembly of the Golgi structure into ministacks, resulting in cleavage and secretion of GOLPH2. CONCLUSIONS: This study demonstrates that GOLPH2 may be a useful tissue biomarker for oesophageal disease. We provide a novel mechanistic insight into the aetiology of oesophageal cancer and reveal novel functions for GOLPH2 in regulating tumour cell migration and invasion, important functions for the metastatic process in oesophageal cancer.

The diagnostic accuracy of endoscopic ultrasound in suspected biliary obstruction and its impact on endoscopic retrograde cholangiopancreatography burden in real clinical practice: a consecutive analysis.

BACKGROUND AND AIMS: Performing endoscopic ultrasound (EUS) before endoscopic retrograde cholangiopancreatography (ERCP) has been described to be useful in cases of suspected biliary obstruction where EUS can triage patients for ERCP. We aimed to determine the diagnostic accuracy of EUS and its impact on ERCP burden in real clinical practice. We also evaluated the safety and efficacy of EUS+ERCP in a single endoscopic session. PATIENTS AND METHODS: Four hundred and eighteen consecutive patients with suspected but unexplained biliary obstruction referred for EUS before possible ERCP were evaluated. The diagnostic accuracy of EUS and its value in predicting the need for ERCP were determined. EUS established whether pancreaticobiliary disorder (PBD) was present and whether therapeutic ERCP was required. These decisions were matched with ERCP findings, histology, clinical course, and follow-up. Where ERCP was indicated, it was performed in the same endoscopic session. RESULTS: EUS was performed in 412/418 patients (feasibility 98.5%), and ERCP was considered necessary in 64% (ERCP avoided in 36%). The single-session EUS and ERCP was safe and effective (264 patients). The diagnostic accuracy of EUS was as follows: choledocholithiasis 99%, malignant strictures 90%, and benign strictures 92%. EUS showed pathology in 42% of patients who had a nondilated biliary system at initial investigations. When EUS indicated a normal common bile duct (n=119), this had a 100% positive predictive value for non-necessity for ERCP. The median overall follow-up period was 12 months (range 6-34 months). CONCLUSION: EUS demonstrated high diagnostic accuracy in this mixed group of PBD. This accurately guided ERCP need and avoided unnecessary ERCP in 36%. EUS and ERCP in the same endoscopic session for the evaluation and management of PBD is technically feasible, with safety and efficacy profiles equivalent to that of each procedure performed independently in different sessions.

Inherited Syndromes Predisposing to Inflammation and GI Cancer.

Cancers arising within the gastrointestinal (GI) tract are commonly associated with an immune component at their inception and later in their maintenance. While many of the immune factors and immune cell types surrounding these lesions have been highlighted, the underlying pre-dispositions in immunesupported carcinogenesis are not well characterised. Inherited Mendelian GI disorders such as polyposis syndromes, while classically due to germline mutations in non-immune genes, commonly demonstrate alterations in key immune and inflammatory genes. In some cases immune based therapies have been shown to provide at least some benefit in animal models of these syndromes. The advent of genome wide association studies has begun to powerfully examine the genetic nature of complex non-Mendelian GI diseases highlighting polymorphisms within immune related genes and their potential to provide the niche in which GI cancers may originate. Here in the role in which Mendelian and non-Mendelian genetics of immune related factors supporting GI malignancy will be presented and discussed.

An integrative genomic approach in oesophageal cells identifies TRB3 as a bile acid responsive gene, downregulated in Barrett's oesophagus, which regulates NF-kappaB activation and cytokine levels.

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.