The Gb3-synthase A4GALT is an epigenetically regulated driver of tumor invasiveness in gastrointestinal cancer

Tumor lipid metabolism has emerged as a critical, yet underexplored, determinant of cancer progression with clinical and prognostic importance. A membrane lipid species of particular interest is globotriaosylceramide (Gb3/CD77), a glycosphingolipid that serves as cellular receptor for the bacterial Shiga toxins, and is upregulated in various malignancies. While preclinical studies have suggested a pro-tumorigenic role for Gb3, the regulatory drivers of its biosynthesis in human tumors have remained elusive. The glycosyltransferase A4GALT, responsible for Gb3 biosynthesis, and the degrading enzyme α-galactosidase A (α-GLA) are two essential molecular determinants of Gb3 cell surface expression. Expression of these enzymes was analyzed on mRNA level in tissues from colorectal cancer, published datasets from gastric, pancreatic, esophageal and colorectal cancer (1213 patients) and in human cell lines. Pharmacological manipulation in vitro using the hypomethylating agent 5-Aza-2-deoxycytidine and histone deacetylase (HDAC) inhibitors induced A4GALT expression in DLD1 colon cancer cells and Gb3 biosynthesis. A4GALT deficiency was induced by CRISPR-Cas9 mutagenesis in human HCT116 colon cancer cells, and its putative effects tested for proliferation, cell migration and invasion. A4GALT deficiency in HCT116 cells confirmed its essential role for Gb3 biosynthesis, leading to resistance against Shiga toxin 1a, and to reduced cancer cell migration and invasion. Gene enrichment analyses revealed that high A4GALT and low α-GLA expression is associated with a distinct gene expression program in gastric, pancreatic and colorectal cancer, including increased signatures of epithelial–mesenchymal transition. A4GALT expression is regulated by chromatin accessibility and DNA methylation at a defined intronic enhancer. In accordance, pan‑cancer analysis of TCGA datasets validated the A4GALT‑high/α‑GLA‑low signature as a negative prognostic indicator across gastrointestinal tumor entities. Our study uncovers an epigenetically regulated lipid metabolic axis involving A4GALT and Gb3 that contributes to aggressive tumor behavior. Of note, this pathway may be therapeutically targetable using natural or synthetic Shiga toxin B-subunit derivatives.