A reflux linked GATA factor fulcrum dictates lineage commitment through GPRC5B during the esophageal dysplastic transition.

Objective: GATA family transcription factors are somatically variable (SV) in esophageal adenocarcinomas (EAC) and inducible by simulated reflux. Our study examines the mechanisms whereby GATA family members, GATA4, GATA6 and the atypical TRPS-1, influence oncogenesis during the Barrett's esophagus (BE) metaplasia-dysplasia transition preceding EAC.

Methods: RNAseq analyses of esophageal cell lines and lesion-derived adult stem cells (ASCs) in conjunction shRNA- or CRISPR-facilitated gene silencing, together with re-analysis of The Cancer Genome Atlas data (TCGA), spatial transcriptomics and organ-on-a-chip (OOAC) studies were employed.

Results: While a GERD history positively correlated with GATA4/6 SV and a columnar-associated gene signature (ANPEP/GATA4) in TCGA EAC cases, it negatively associated with a squamous lineage-linked signature (TP63/SOX15) containing TRPS1. In experimental data, opposing effects on regulators of squamous and columnar lineage identity were uncovered between TRPS1 and classical GATA factors (GATA4/6). Interrogation of this GATA "fulcrum" defined further genes (CGN, IL6R and GPRC5B) targeted for TRPS1-mediated suppression or GATA4/6 activation. A novel spatial transcriptomic signature of BE-associated high-grade-dysplasia (HGD) captured GATA fulcrum action through GPRC5B expression. Functionally, GPRC5B was found to be low-pH-responsive, to increase proliferative and colony formation rates, and when over-expressed facilitate a hyper proliferative HGD-like transformation of BE-ASCs. Using an OOAC platform, cellular overgrowth, reduced luminal villus structures, lower goblet cell numbers, and loss of intestine-associated marker gene expression (TFF3/MUC2), were observed following GPRC5B over-expression in BE-ASCs, mirroring HGD.

Conclusions: This study identifies critical GATA factor-mediated processes underlying cellular phenotype in the BE-HGD-EAC transition and identifies GATA-inducible GPRC5B as a functional marker and possible driver of progression through HGD to EAC.