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

Abstract

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.

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

“…Sample size for messenger RNA (mRNA) expression analysis of immune-related factors in BE (N = 17) and EAC (N = 23) patients and controls (N = 17) was determined through previous use of this clinical cohort in published reports. 25 Written, oral, and informed consent was provided by all patients who contributed tissue biopsy specimens for gene expression aspects of this study. Ethical approval for this study was granted by the Research Ethics Committee of Tallaght Hospital and St. James’ Hospital (Dublin, Ireland) and performed in accordance with the associated guidelines.…”

“…Proteins separated by SDS-PAGE were transferred to a polyvinylidene difluoride membrane in semidry conditions in a power blotter (Pierce, Thermo Fischer) as previously described. 18 , 19 , 22 , 25 Membranes were collected in PBS followed by incubation in milk protein–based blocking solution for 30 minutes. Primary antibodies to LIF, C1QA (ab108325; Abcam), TREM2 (Sigma), TYROBP or DAP12 (ab124834; Abcam), spleen tyrosine kinase (SYK) (D3Z1E; Cell Signaling), and p-SYK (Cell Signaling) were diluted in blocking solution at 1:1000, 1:500, 1:250, 1:300, 1:300, and 1:200, respectively, and incubated with the appropriate membranes for 24 hours at 4°C followed by washing with PBS-Tween and further room temperature incubation with appropriate secondary horseradish-peroxidase–conjugated antibody at a 1:2000 dilution for 2 hours.…”

“…Data sets derived from Kim et al, 29 Kimchi et al, 30 and Ostrowski et al 31 were re-analyzed in Genespring GX (Agilent, Santa Clara, CA) as described previously. 25 The expression of genes whose siRNA-mediated silencing resulted in greater than 50% cell death and a Z score level <-1.645 were examined in the gene expression microarray (GEM) data sets of BE and EAC tissues. These GEM studies were analyzed at a Mann–Whitney significance level of P < .001.…”

“…As outlined in previous publications, 18 , 25 gene expression analysis was performed by real-time relative reverse-transcription polymerase chain reaction using a 7900HT thermocycler with predesigned real-time primers and probes (Applied Biosystems, Foster City, CA). Relative fold changes in mRNA expression levels were calculated using the delta delta Ct method and glyceraldehyde-3-phosphate dehydrogenase as the denominator control gene.…”

“…Nonparametric Mann–Whitney testing was used for analysis of significance in real-time gene expression experiments performed with both biological (N = 3–23) and technical replicates (N = 3). 18 , 19 , 25 The performance of high-throughput screening was determined through the use of a Z-factor quality metric. 34 Statistical interpretation of siRNA screening results was achieved through Z score ranking of normalized median values.…”