REGULATION OF STAT3 SIGNALING BY PROTEIN ADDUCTION IN THE EARLY STAGES OF ESOPHAGEAL ADENOCARCINOMA

Background: The most common risk factor for esophageal adenocarcinoma is gastroesophageal reflux disease (GERD), a digestive disorder that affects 20% of adults in North America. The exposure of esophageal cells to chronic gastroesophageal reflux induces STAT3 signaling that promotes esophageal tumorigenesis through the activation of pro-inflammatory, pro-survival and anti-apoptotic signaling pathways. In this study, we focused on the novel mechanisms of STAT3 regulation by protein adduction in conditions of esophageal reflux.
Methods and Results: For the first time, in addition to the known canonical pathways, we found that the protein modification and subsequent adduction of STAT3 protein activates its biological function. The Isolevuglandins (IsoLGs) that belongs to the family of γ-ketoaldehydes are involved in this process. IsoLGs are formed through free radical or ROS and enzymatic cyclooxygenation of polyunsaturated fatty acids and are highly reactive to free amines on lysine residues forming protein adducts. The adduction of multiple proteins by isoLGs was found in the esophagus of GERD patients and surgical mice model with esophagojejunostomy that recapitulates the human GERD conditions. The STAT3 protein was adducted with IsoLGs in the esophageal cells in conditions of oxidative stress induced by the treatment with acidic bile salts (ABS) that mimic the constituents of gastroesophageal reflux. Investigating the consequence of STAT3 adduction using focus array (84 key genes of human STAT3 signaling pathway), qPCR, Western blotting and Chromatin immunoprecipitation sequencing (ChIP-seq), showed that the modification of STAT3 by isoLGs increases the genome-wide binding of STAT3 protein to the promoter of its target genes causing activation of STAT3-dependent transcription. Using immunofluorescence, immunoprecipitation, ubiquitination assay and posttranslational modification analyzes, we found that the isoLG-STAT3 adduction increases the stability and activity of STAT3 protein in conditions of reflux. We also tested the isoLG scavengers, such as 2-hydroxybenzylamine (2-HOBA), and found that these compounds are able to prevent the activation of STAT3 both in vitro and in vivo.
Conclusions: Combined, our studies revealed a novel mechanism of STAT3 protein adduction that activates STAT3 signaling in the preneoplastic stages of esophageal cancer. Our findings pave the way for the development of new cancer chemopreventive drugs.