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

Abstract

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.

High Content Imaging of Barrett’s-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions

“…An siRNA screening protocol ( Figure 1 A ) using a cell line representing Barrett’s-associated high-grade dysplasia, CP-D BE-HGD cells, a druggable genome siRNA library consisting of 6033 siRNA pools and HCA of cytoskeletal proteins (F-actin, α-tubulin, and Hoechst) was used as previously described. 30 To reduce data dimensionality (8 images per well, 7000 wells, 4 channels), initial analysis and data transformations of screening images was performed using a singular analytic parameter measuring the difference between the overall area of the cell (F-actin) and that of tubulin staining (A minus T [A-T] parameter), thus reflecting extensions, contractions, or bundling associated with cellular transitions. Locally weighted polynomial regression normalization first was performed to account for cell shape changes as a consequence of alterations in cell density that occur as a result of gene loss-mediated cell death, growth inhibition, or regional variations ( Figure 1 B ).…”

“…As previously described, siRNA siGenome-SMARTpool (Dharmacon, GE Healthcare, Horizon Discovery, Waterbeach, UK) were used for initial screening followed by siON-TARGETplus build (Dharmacon) in all other validation experiments as an alternate verification strategy. 30 siRNAs were delivered to cells using Dharmafect transfection reagent (DF1-4; Dharmacon) in a reverse-transfection protocol with a cell culture media change at 24 hours after transfection. Validation of siRNA knockdown was achieved by Western blot and real-time reverse-transcription polymerase chain reaction for respective proteins (N = 3 biological replicates in 2 cell lines) and mRNAs (N = 3 technical and biological replicates) at 72–96 hours after transfection with siRNA.…”

Esophageal Adenocarcinomas: A Need for Speed Driven by Immune Pathways That Have Druggable Targets

“…As we move toward achieving our goal of finding better treatments for EAC patients, a better understanding of tumor biology is critical if we are to expose key cellular vulnerabilities that might be exploited for molecular targeting. The immune addiction of EACs shown by Duggan et al 2 is one such vulnerability, and we would be happy to help EACs kick their habit!…”

“…In this issue of Cellular and Molecular Gastroenterology and Hepatology , Duggan et al 2 described their use of a loss-of-function screening approach to identify potential therapeutic targets for EAC. First, they studied the effects of a druggable genome small interfering RNA (siRNA) library (>6000 individual gene-targeting siRNA pools) on the viability of a human high-grade dysplastic Barrett’s epithelial cell line (CPD).…”

“…The elegant proof-of-principle studies by Duggan et al 2 have shown the power of integrating functional genomic studies in cell lines with genetic data from patients to provide an unbiased avenue for identifying novel, druggable therapeutic targets. In addition, these studies have shown new insights into how EACs appear to be addicted to an inflammatory-immune drive.…”