The Receptor for Advanced Glycation End Products (RAGE) prompts the motility of breast cancer cells through EphA3

Abstract

The receptor for advanced glycation-end products (RAGE) and its ligands have been associated with obesity, inflammation and diabetes. Recently, several studies showed that RAGE signaling may contribute to the metastatic progression of breast cancer (BC), although the mechanisms underlined remain to be fully characterized. Here, we aimed at defining the molecular events by which RAGE may prompt aggressive features in estrogen receptor (ER)-positive BC cells. As model system, we engineered human MCF7 and T47D BC cells to stably overexpress RAGE. The RAGE-mediated changes in cell morphology, migration, invasion and colony formation were evaluated in vitro through scanning electron microscopy, migration, invasion and clonogenic assays, while in vivo effects were explored in zebrafish xenografts models. Importantly, high-throughput RNA sequencing allowed us to analyze the transcriptomic landscape of RAGE-overexpressing BC cells. Thereafter, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to recognize the biological functions of the differentially expressed genes (DEGs) identified in RAGE-overexpressing respect to wild type BC cells. Molecular biology techniques, including flow cytometry, real-time PCR, chromatin immunoprecipitation, immunofluorescence and Western blot assays, were employed to investigate the molecular mechanisms implicated in the regulation of a novel RAGE target gene named EphA3. The clinical significance of EphA3 was explored in the TCGA cohort of BC patients through the survivALL package, whereas the involvement of EphA3 in mediating migratory and invasive effects was ascertained in both BC cells and important component of the tumor microenvironment like cancer-associated fibroblasts (CAFs). Our findings revealed that RAGE-overexpressing BC cells exhibit an enhanced dissemination potential respect to wild type BC cells. Moreover, we have found that EphA3 may act as a main mediator of the RAGE-induced motility of BC cells and CAFs. Overall, our findings suggest that EphA3 may be considered as a novel RAGE target gene that may prompt BC cell migration and invasion. Therefore, these data highlight the potential significance of targeting RAGE and EphA3 in the treatment of BC, particularly in diabetic or obese patients that show high RAGE levels.